Crystalline forms of genistein

ABSTRACT

The disclosure relates to new crystalline forms of genistein. The disclosed crystalline forms include crystalline genistein sodium salt dihydrate; crystalline genistein potassium salt dihydrate; crystalline genistein calcium salt; crystalline genistein magnesium salt; crystalline genistein L-lysine salt; crystalline genistein N-methylglucamine salt; crystalline genistein N-ethylglucamine salt; crystalline genistein diethylamine salt; and crystalline genistein monohydrate. The disclosure also relates to the novel genistein salts represented by these crystalline forms. Therapeutic compositions containing at least one of these crystalline forms of genistein and/or a genistein salt and a pharmaceutically acceptable carrier are described. The disclosure also relates to methods of treating cancer comprising the step of administering to a patient in need thereof a therapeutically effective amount of a therapeutic composition containing the compounds of the disclosure, of a crystalline form of genistein, or of a genistein salt.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application61/121,778, filed Dec. 11, 2008, and to U.S. provisional application61/121,787, filed Dec. 11, 2008, both of which are incorporated hereinby reference.

BACKGROUND

Cancer is characterized by uncontrolled cell growth which occurs whenthe normal regulation of cell proliferation is lost. This loss oftenappears to be the result of dysregulation of the cellular pathwaysinvolved in cell growth and division, apoptosis, angiogenesis, tumorinvasion and metastasis.

Genistein,4′5,7-trihydroxyisoflavone-5,7-dihydro-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one,(shown below), is a natural compound present in plants such as soy.Genistein's potential role in the prevention and treatment of a numberof human diseases

including cancer has been extensively studied. Genistein is a BCS classII isoflavone that is commercially available from a number of sourcesincluding LC Laboratories, Woburn, Mass. The cellular targets forgenistein and the signaling pathways regulated by genistein have beenidentified and those related to cancer include targets and pathwaysimportant for cell growth and division, apoptosis, angiogenesis, tumorinvasion and metastasis. In addition to the inherent anti-tumor effectsof genistein itself, studies have shown that genistein also potentiates,or accentuates, the anti-tumor effects of several clinically usedchemotherapeutic agents both in vitro in human cancer cell lines and invivo in animal models of cancer. From a therapeutic perspective, thesedata are interesting as chemotherapy is the cornerstone in the treatmentof most solid tumors.

Genistein is practically insoluble in water but has high cell membranepermeability. Low water solubility and slow dissolution rate are oftenlimiting factors responsible for the low bioavailability ofpharmaceutical compounds, limiting their application.

Despite the long known fact that genistein has certain properties ofanti-cancer drugs, no successful genistein treatment regimens have been,or are, employed in the treatment of cancers. One plausible explanationfor this is probably the poor solubility and poor bioavailability aswell as the rapid phase II metabolism of genistein in its known form.

Due to the development of the drug discovery strategy over the last 20years, physicochemical properties of drug development candidates havechanged significantly. The development candidates are generally morelipophilic and less water soluble, which creates huge problems for theindustry. Research has shown that some drug candidates fail in theclinical phase due to poor human bioavailability and problems with theformulation. Traditional methods to address these problems, withoutcompletely redesigning the molecule, include salt selection, producingamorphous material, particle size reduction, pro-drugs, and differentformulation approaches. Recently, crystalline forms of activepharmaceutical ingredient (API) have been used to alter thephysicochemical properties of the API.

Although therapeutic efficacy is the primary concern for a therapeuticagent, the salt and solid state form (i.e., the crystalline or amorphousform) of a drug candidate can be critical to its pharmacologicalproperties and to its development as a viable API. For example, eachsalt or each crystalline form of a drug candidate can have differentsolid state (physical and chemical) properties. The differences inphysical properties exhibited by a novel solid form of an API (such as acocrystal, salt, or polymorph of the original compound) affectpharmaceutical parameters such as storage stability, compressibility anddensity (important in formulation and product manufacturing), andsolubility and dissolution rates (important factors in determiningbioavailability). Because these practical physical properties areinfluenced by the solid state properties of the crystalline form of theAPI, they can significantly impact the selection of a compound as anAPI, the ultimate pharmaceutical dosage form, the optimization ofmanufacturing processes, and absorption in the body. Moreover, findingthe most adequate polymorphic form for further drug development canreduce the time and the cost of that development.

Obtaining crystalline forms of an API is extremely useful in drugdevelopment. It permits better characterization of the drug candidate'schemical and physical properties. It is also possible to achieve desiredproperties of a particular API by forming a salt of the API and/or acrystalline salt of the API, Crystalline forms and crystalline saltsoften have better chemical and physical properties than the free base inits amorphous state. Such salts and crystalline forms may, as with thepresent invention, possess more favorable pharmaceutical andpharmacological properties or be easier to process than the amorphouspolymorphic form. They may also have better storage stability.

One such physical property, which can affect processability, is theflowability of the solid, before and after milling, Flowability affectsthe ease with which the material is handled during processing into apharmaceutical composition. When particles of the powdered compound donot flow past each other easily, a formulation specialist must take thatfact into account in developing a tablet or capsule formulation, whichmay necessitate the use of glidants such as colloidal silicon dioxide,talc, starch or tribasic calcium phosphate.

Another potentially important solid state property of an API is itsdissolution rate in aqueous fluid. The rate of dissolution of an activeingredient in a patient's stomach fluid may have therapeuticconsequences since it impacts the rate at which an orally administeredactive ingredient may reach the patient's bloodstream.

By forming and/or crystallizing a salt of an API, a new solid state formof the API may have unique properties compared with existing solid formsof the API or its salt. For example, a crystalline salt may havedifferent dissolution and solubility properties than the API itself andcan be used to deliver APIs therapeutically. New drug formulationscomprising crystalline salts of APIs may have superior properties overexisting drug formulations.

A crystalline salt or other crystalline form of an API generallypossesses distinct crystallographic and spectroscopic properties whencompared to other forms having the same chemical composition.Crystallographic and spectroscopic properties of the particular form aretypically measured by X-ray powder diffraction (XRPD) and single crystalX-ray crystallography, among other techniques. Particular crystallineforms often also exhibit distinct thermal behavior. Thermal behavior ismeasured in the laboratory by such techniques as capillary meltingpoint, thermogravimetric analysis (TGA) and differential scanningcalorimetry (DSC).

SUMMARY

The invention relates to crystalline forms of genistein, includingcrystalline genistein salts and a crystalline genistein hydrate.Therapeutic compositions containing the crystalline forms of genisteinof the invention represent another embodiment of the invention, as domethods of treating or preventing cancer and other hyperproliferativediseases with those crystalline forms of the invention or therapeuticcompositions containing them. Therapeutic compositions of crystallinegenistein may also be used for the treatment or prevention of chronicinflammation, infection, cystic fibrosis and amyloidosis. As used hereinand as known in the art, the term “ambient temperature” means atemperature within an enclosed space at which humans are accustomed,i.e., room temperature. For example, ambient temperature may range, forexample, from about 20° C. to about 25° C.

As used herein and as known in the art, the term “approximately” meansnear to in quantity or amount.

As used herein and as known in the art, the term “slurry” means asuspension of solids in a liquid.

As used herein and as known in the art, the term “°2θ” isinterchangeable with [degree-two-theta], [°2Th.], and variationsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures, which are described below and which areincorporated in and constitute a part of the specification, illustrateexemplary embodiments according to the disclosure and are not to beconsidered limiting of the scope of the invention, for the invention mayadmit to other equally effective embodiments. The figures are notnecessarily to scale, and certain features and certain views of thefigures may be shown exaggerated in scale or in schematic in theinterest of clarity and conciseness.

FIG. 1 depicts an XRPD pattern of crystalline genistein sodium saltdihydrate.

FIG. 2 depicts a DSC trace of dried crystalline genistein sodium saltdihydrate.

FIG. 3 depicts a gravimetric vapor sorption (GVS) trace of crystallinegenistein sodium salt dihydrate.

FIG. 4 depicts a TGA trace from a sample of prepared crystallinegenistein sodium salt dihydrate that was dried at ambient temperaturefor about 24 hours.

FIG. 5 depicts a TGA trace from a sample of prepared crystallinegenistein sodium salt dihydrate that was dried at 80° C. overnight.

FIG. 6 depicts four XRPD patterns of crystalline genistein sodium saltdihydrate taken after stability studies at 80° C. for 7 days and at 40°C./75 relative humidity (RH) % for 7 days.

FIG. 7 is a ¹H nuclear magnetic resonance (NMR) spectra of crystallinegenistein sodium salt dihydrate.

FIG. 8 depicts XRPD patterns after a hydration study of crystallinegenistein sodium salt dihydrate.

FIG. 9 is a molecular model of crystalline genistein sodium saltdihydrate, illustrating the centrosymmetric disodium cation in thedimeric structure of crystalline genistein sodium salt dihydrate,wherein the intramolecular hydrogen bonds are shown as dashed lines.

FIG. 10 is a molecular model illustrating a layer formation ofcrystalline genistein sodium salt dihydrate.

FIG. 11 is a molecular model illustrating a packing of crystallinegenistein sodium salt dihydrate.

FIG. 12 depicts a calculated XRPD pattern based on single crystal datafor crystalline genistein sodium salt dihydrate.

FIG. 13 depicts the plasma concentration of total genistein afterintraduodenal administration of genistein and crystalline genisteinsodium salt dihydrate (mean, n=3).

FIG. 14 depicts an XRPD pattern for crystalline genistein sodium saltdihydrate from the large scale synthesis.

FIG. 15 depicts an XRPD pattern for amorphous genistein potassium salt.

FIG. 16 depicts an XRPD pattern for crystalline genistein potassium saltdihydrate.

FIG. 17 depicts a TGA trace of crystalline genistein potassium saltdihydrate.

FIG. 18 depicts a DSC trace of crystalline genistein potassium saltdihydrate.

FIG. 19 depicts a GVS trace of crystalline genistein potassium saltdihydrate.

FIG. 20 depicts the ¹H NMR of crystalline genistein potassium saltdihydrate.

FIG. 21 depicts XRPD patterns from the stability study of crystallinegenistein potassium salt dihydrate.

FIG. 22 depicts XRPD patterns for a hydration study of crystallinegenistein potassium dihydrate.

FIG. 23 depicts an XRPD pattern for crystalline genistein calcium salt.

FIG. 24 depicts the TGA trace for crystalline genistein calcium salt.

FIG. 25 depicts an XRPD pattern for crystalline genistein magnesiumsalt, 1 equivalent.

FIG. 26 depicts the TGA trace for crystalline genistein magnesium salt,1 equivalent.

FIG. 27 depicts an XRPD pattern for crystalline genistein magnesiumsalt, 2 equivalents.

FIG. 28 depicts the TGA trace for crystalline genistein magnesium salt,2 equivalents.

FIG. 29 depicts an XRPD pattern for crystalline genistein.

FIG. 30 depicts an XRPD pattern for crystalline genistein L-Lysine saltfrom toluene.

FIG. 31 depicts an XRPD pattern for crystalline genistein L-Lysine saltfrom isopropanol.

FIG. 32 depicts the TGA trace for a crystalline genistein/genisteinmixture from isopropanol.

FIG. 33 depicts an XRPD pattern for crystalline genisteinN-methylglucamine (meglumine) salt.

FIG. 34 depicts an XRPD pattern for crystalline genisteinN-ethylglucamine (eglumine) salt, prepared from acetone.

FIG. 35 depicts an XRPD pattern for crystalline genisteinN-ethylgiucamine (eglumine) salt, prepared from isopropanol.

FIG. 36 depicts a TGA trace of crystalline genistein N-ethylglucaminesalt from acetone.

FIG. 37 depicts an XRPD pattern for crystalline genistein diethylaminesalt.

FIG. 38 depicts an XRPD pattern for crystalline genistein monohydrate.

FIG. 39 depicts a TGA trace of crystalline genistein monohydrate.

DETAILED DESCRIPTION

The current invention relates to improvements of the physiochemicalproperties of genistein, whereby this compound may be suitable for drugdevelopment. Disclosed herein are several new crystalline forms ofgenistein, including, for example, crystalline genistein salts ofsodium, potassium, magnesium, N-methylglucamine (meglumine), calcium,L-lysine, N-ethylgiucamine (eglumine), and diethylamine, as well as acrystalline monohydrate of genistein. These crystalline forms ofgenistein are described below. Although crystalline forms of genisteinare described herein, the invention also relates to novel chemicalcompositions containing the disclosed crystalline forms of genistein.The therapeutic uses of those crystalline forms are described as well astherapeutic compositions containing them. The methods used tocharacterize the crystalline forms are also described below.

One embodiment of the invention relates to a crystalline genisteinsodium salt dihydrate. The crystalline genistein sodium salt dihydratemay possess suitable characteristics for pharmaceutical development. Theonly possible negative may be its needle-like morphology which is notnecessarily ideal for flowability or compression during manufacture. Theneedle-like morphology was observed using Polarized Light Microscopy(PLM). Milling of this crystalline needle-like material, or similartechniques known in the art, may be used to achieve more uniformparticle morphology, which may be used to prepare the material formanufacturing its pharmaceutical composition. One of ordinary skill candetermine particle sizes appropriate for a desired pharmaceuticalcomposition, Particle sizes of about 5 μm, for example; may be used. Itshould be noted however, that sustained milling may dehydrate thematerial due to the high temperatures involved during such processes. Onthe other hand, the 80° C. storage tests have indicated that thematerial can exist as a hydrate at elevated temperatures over a 7 dayperiod with only a slight change. This mitigates the risk of dehydrationon milling.

As shown in FIG. 9, the crystalline genistein sodium salt dihydrate ofthe invention has a dimeric structure centrosymmetric disodium cation inassociation with two genistein molecules and four water molecules. Thecrystalline genistein sodium salt dihydrate may be prepared from, forexample, IPA (isopropanol or propan-2-ol), a common solvent, at ambienttemperature without the need for any special treatment such astemperature cycling, sonication or rapid evaporation. As shown inExample 1 below, the crystalline genistein sodium salt dihydrate of theinvention possesses excellent stability. It is more soluble in water,aqueous solvent systems and organic solvents than genistein. Inaddition, the crystalline genistein sodium salt dihydrate shows superiorearly and late intrinsic kinetic solubility profiles as compared togenistein. The crystalline genistein sodium dihydrate of the inventionhas also been shown to have greater bioavailability than genistein.

Another embodiment of the invention is crystalline genistein potassiumsalt dihydrate. The crystalline genistein potassium salt dihydrate mayalso be prepared from, for example, IPA (isopropanol or propan-2-ol), acommon solvent, at ambient temperature without the need for any specialtreatment such as temperature cycling, sonication or rapid evaporation.Crystalline genistein potassium salt dihydrate readily forms from solidgenistein potassium salt. Genistein potassium salt appears to be anunstable anhydrous amorphous salt at the point of recovery, which thenrapidly absorbs water from the surroundings to crystallize into thedihydrated material. As discussed in Example 2 below, the crystallinegenistein potassium dihydrate salt has good stability. The genisteinpotassium salt dihydrate is crystalline and has a needle-like morphology(but thicker needles than the corresponding crystalline genistein sodiumsalt dihydrate).

In addition to the crystalline sodium and potassium genistein salts ofthe invention, other separate embodiments of the invention relate tocrystalline salts of genistein with magnesium, N-methylglucamine(meglumine), calcium, L-lysine, N-ethylglucamine (eglumine anddiethylamine. A further embodiment of the invention relates to acrystalline monohydrate form of genistein. Each of these crystallineforms of genistein, their preparation and characterization, aredescribed in the examples below,

Therapeutic Uses of the Crystalline Forms of Genistein

The invention relates to therapeutic uses of at least one crystallineform of genistein, for example, at least one crystalline genistein salt.The term “treatment” or “treating” means any treatment of a disease ordisorder in a mammal, including, inhibiting the disease or disorder,that is, arresting or suppressing the development of clinical symptoms;and/or relieving the disease or disorder, that is, causing theregression of clinical symptoms.

The crystalline forms of genistein according to the invention may beuseful as a medicament, which may be used to treat hyperproliferativediseases such as, for example, various cancers, including, for example,colorectal, gastric, esophageal, breast, lung, prostate, bladder, brain,renal, ovarian, liver, skin, thyroid, and pancreatic cancer, as well asleukemias or lymphomas. The leukemias and lymphomas mentioned herein maybe tumors of myeloid lineage such as, for example, acute myeloidleukemia or of lymphoid lineage.

Additionally, the crystalline forms of genistein disclosed herein mayalso be used in a method of treatment of a warm-blooded animal such as,for example, man, by therapy. For example, a crystalline genistein saltaccording to the invention may be useful in a method of treatment ofhyperproliferative diseases such as, for example, various cancers,including, for example, colorectal, gastric, esophageal, breast, lung,prostate, bladder, brain, renal, ovarian, liver, skin, thyroid, andpancreatic cancer, as well as leukemias or lymphomas. The leukemias andlymphomas mentioned herein may be tumors of myeloid lineage such as, forexample, acute myeloid leukemia or of lymphoid lineage.

Moreover, crystalline forms of genistein according to the invention maybe used in the method of treating a human suffering from ahyperproliferative diseases such as, for example, various cancers,including, for example, colorectal, gastric, esophageal, breast, lung,prostate, bladder, brain, renal, ovarian, liver, skin, thyroid, andpancreatic cancer, as well as leukemias or lymphomas. In anotherembodiment, the crystalline forms of genistein according to thedisclosure may be used to prevent hyperproliferative diseases such as,for example, various cancers, including, for example, colorectal,gastric, esophageal, breast, lung, prostate, bladder, brain, renal,ovarian, liver, skin, thyroid, and pancreatic cancer, as well asleukemias or lymphomas. The leukemias and lymphomas mentioned herein maybe tumors of myeloid lineage, such as, for example, acute myeloidleukemia or of lymphoid lineage, comprising the steps of administeringto a person in need thereof a therapeutically effective amount of atleast one crystalline form of genistein. The use of at least onecrystalline form of genistein in any of the methods of treating a humandescribed above also form aspects of this invention.

The treatment defined herein may be applied as a sole therapy or mayinvolve, in addition to the at least one compound of the invention,conventional surgery or radiotherapy or chemotherapy. Such chemotherapymay include one or more of the following categories of anti-tumouragents: (i) antiproliferative/antineoplastic drugs and combinationsthereof, as used in medical oncology, such as alkylating and alkylatinglike agents (for example, cis-platin, carboplatin, cyclophosphamide,nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas),antimetabolites (for example, gemcitabine HCl, 5-fluorouracil, tegafur,raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea),antitumour antibiotics (for example, anthracyclines like adriamycin,bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin), antimitotic agents (for example, vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere), and topoisomerase inhibitors (forexample, epipodophyllotoxins like etoposide and teniposide, amsacrine,topotecan and camptothecin); (ii) cytostatic agents, such asantioestrogens (for example, tamoxifen, toremifene, raloxifene,droloxifene and iodoxyfene), oestrogen receptor antagonists (for examplefulvestrant), antiandrogens (for example, bicalutamide, flutamide,nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists(for example, goserelin, leuprorelin and buserelin), progestogens (forexample, megestrol acetate), aromatase inhibitors (for example,anastrozole, letrozole, vorazole and exemestane), and inhibitors of5-alpha-reductase (for example, finasteride); (iii) agents which inhibitcancer cell invasion (for example, metalloproteinase inhibitors likemarimastat and inhibitors of urokinase plasminogen activator receptorfunction); (iv) inhibitors of growth factor function for example, suchinhibitors include growth factor antibodies, growth factor receptorantibodies (for example, the anti-ErbB2 antibody trastuzumab(Herceptin), and the anti-ErbB1 antibody (cetuximab)), farnesyltransferase inhibitors, tyrosine kinase inhibitors, and serine-threoninekinase inhibitors, for example, inhibitors of the epidermal growthfactor family (for example, EGFR family tyrosine kinase inhibitors suchasN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, AZDI 839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)qumazolin-4-amine(CI 1030; inhibitors of the platelet-derived growth factor family, andinhibitors of the hepatocyte growth factor family; (v) antiangiogenicagents such as those which inhibit the effects of vascular endothelialgrowth factor (for example, the anti-vascular endothelial cell growthfactor antibody bevacizumab (Avastin) and compounds such as thosedisclosed in International Patent Applications WO 97/22596, WO 97/30035,WO 97/32856 and WO 98/13354) and compounds that work by other mechanisms(for example, linomide, inhibitors of integrin function andangiostatin); (vi) vascular damaging agents such as Combretastatin A4and compounds disclosed in International Patent Applications WO99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO02/08213; (vii) antisense therapies, for example, those which aredirected to the targets listed above, such as ISIS 2503, an anti-rasantisense; (viii) gene therapy approaches, including, for example,approaches to replace aberrant genes such as aberrant p53 or aberrantBRCAI BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approachessuch as those using cytosine deaminase, thymidine kinase or a bacterialnitroreductase enzyme and approaches to increase patient tolerance tochemotherapy or radiotherapy such as multi-drug resistance gene therapy;and (ix) immunotherapy approaches, including for example ex-vivo and invivo approaches to increase the immunogenicity of patient tumour cells,such as transfection with cytokines such as interleukin 2, interleukin 4or granulocyte-macrophage colony stimulating factor, approaches usingtransfected immune cells such as cytokine-transfected dendritic cells,approaches using cytokine-transfected tumor cell lines, and approachesusing anti-idiotypic antibodies.

In the treatment discussed above, at least one crystalline form ofgenistein according to the invention may also be used in combinationwith one or more cell cycle inhibitors, for example, with cell cycleinhibitors which inhibit cyclin-dependent kinases (CDK), or incombination with imatinib mesylate (Glivec). Such joint treatment may beachieved by way of the simultaneous, sequential or separate dosing ofthe individual components of the treatment. Such combination productsmay employ at least one compound of this invention within the dosagerange described herein and the other at least onepharmaceutically-active agent within its approved dosage range.Combination products may be formulated into a single dosage form.

The invention also provides a combination that may be suitable for usein the treatment of cell proliferative disorders (such as cancer)comprising at least one crystalline form of genistein, such as at leastone crystalline genistein salt, as defined hereinbefore, and at leastone additional anti-tumor agent as defined hereinbefore. Suchcombination may serve as a pharmaceutical product for the conjointtreatment of cell proliferative disorders (such as cancer).

In addition to their use in therapeutic medicine, at least onecrystalline form of genistein according to the invention may also beuseful as pharmacological tools in the development and standardizationof in vitro and in vivo test systems for the evaluation of the effectsof inhibitors of cell cycle activity in laboratory animals such as cats,dogs, rabbits, monkeys, rats and mice, as part of the search for newtherapeutic agents.

Another aspect of invention relates to therapeutic uses of at least onecrystalline form of genistein according to the invention in thepreparation of a medicament for the treatment of a disease where theinhibition of inflammation is beneficial, such as, for example, chronicinflammation, inflammatory bowel disease, Crohn's disease, Sjögren'sdisease, rheumatoid arthritis, arthritis, atopic dermatitis, vasculitis,psoriasis, benign prostate hyperplasia, wound healing, end stage renaldisease, chronic kidney disease, chronic obstructive pulmonary disease,or asthma.

Additionally, at least one crystalline form of genistein according tothe invention may also be used in the preparation of a medicament forthe treatment of a disease where the inhibition of infection isbeneficial, such as, for example, local infection, systemic infection,sepsis, systemic fungal infection, or local fungal infection.

Yet another aspect of the invention relates to the use of at least onecrystalline form of genistein for the treatment of a disease whererestoring normal chloride and salt (water) movements in body organs andpeople's glands is beneficial, such as, for example, stimulating thecystic fibrosis transmembrane conductance regulator.

Yet another aspect of the invention relates to the use of at least onecrystalline form of genistein for the treatment of a disease whereinhibition of a soluble protein from forming insoluble extracellularfibril deposits causing organ dysfunction is beneficial, such as, forexample, inhibition of transthyretin (TTR) amyloidoses caused byalterations in the amino acid sequence of the TTR gene product. Inanother embodiment of the disclosure, at least one crystalline form ofgenistein described herein may be used for the treatment of FamilialAmyloid Polyneuropathy.

Pharmaceutical Compositions Containing Crystalline Forms of Genistein

The invention also relates to pharmaceutical compositions comprising atherapeutically effective amount of at least one crystalline form ofgenistein according to the invention and a pharmaceutically acceptablecarrier (also known as a pharmaceutically acceptable excipient). Asdiscussed above, the crystalline forms of genistein according to theinvention may be therapeutically useful for the treatment or preventionof, for example, the disease states discussed above, including, forexample, those associated with abnormal angiogenesis.

Pharmaceutical compositions for the treatment of those disease statesmay contain a therapeutically effective amount of at least onecrystalline form of genistein according to the invention todown-regulate the transcription of genes involved in controllingangiogenesis for treatment of a patient with the particular disease. Apharmaceutical composition of the invention may be in any pharmaceuticalform which contains at least one crystalline form of genistein accordingto the invention. The pharmaceutical composition may be, for example, atablet, capsule, liquid suspension, injectable, topical, or transdermal.The pharmaceutical compositions generally contain, for example, about 1%to about 99% by weight of at least one crystalline form of genistein ofthe invention and, for example, 99% to 1% by weight of at least onesuitable pharmaceutical excipient. In one embodiment, the compositionmay be between about 5% and about 75% by weight of at least onecrystalline form of genistein of the invention with the rest being atleast one suitable pharmaceutical excipient or at least one otheradjuvant, as discussed below.

A “therapeutically effective amount of at least one crystalline form ofgenistein according to the invention” is generally in the range of about0.05-about 500 mg/kg. The actual amount required for prophylaxis ortreatment of any particular patient may depend upon a variety of factorsincluding, for example, the disease state being treated and itsseverity; the specific pharmaceutical composition employed; the age,body weight, general health, sex and diet of the patient; the mode ofadministration; the time of administration; the route of administration;and the rate of excretion of the crystalline form of genistein; theduration of the treatment; any drugs used in combination or coincidentalwith the specific compound employed; and other such factors well knownin the medical arts. These factors are discussed in Goodman and Gilman's“The Pharmacological Basis of Therapeutics”, Tenth Edition, A. Gilman,J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, whichis incorporated herein by reference. The crystalline forms of genisteinaccording to the invention and pharmaceutical compositions containingthem may be used in combination with anticancer or other agents that aregenerally administered to a patient being treated for cancer. They mayalso be co-formulated with one or more of such agents in a singlepharmaceutical composition.

Depending on the type of pharmaceutical composition, thepharmaceutically acceptable carrier may be chosen from any one or acombination of carriers known in the art. The choice of thepharmaceutically acceptable carrier depends upon the pharmaceutical formand the desired method of administration to be used. For apharmaceutical composition of the invention, that is one having at leastone crystalline form of genistein of the invention, a carrier should bechosen that maintains the crystalline form. In other words, the carriershould not substantially alter the crystalline form of genistein. Norshould the carrier be otherwise incompatible with the crystalline formof genistein salt used, such as by producing any undesirable biologicaleffect or otherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition.

The pharmaceutical compositions of the invention may be prepared bymethods know in the pharmaceutical formulation art, for example, seeRemington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company,Easton, Pa., 1990), which is incorporated herein by reference. In asolid dosage form, at least one crystalline form of genistein may beadmixed with at least one pharmaceutically acceptable excipient such as,for example, sodium citrate or dicalcium phosphate or (a) fillers orextenders, such as, for example, starches, lactose, sucrose, glucose,mannitol, and silicic acid, (b) binders, such as, for example, cellulosederivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose,and gum acacia, (c) humectants, such as, for example, glycerol, (d)disintegrating agents, such as, for example, agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, croscarmellosesodium, complex silicates, and sodium carbonate, (e) solution retarders,such as, for example, paraffin, (f) absorption accelerators, such as,for example, quaternary ammonium compounds, (g) wetting agents, such as,for example, cetyl alcohol, and glycerol monostearate, magnesiumstearate and the like (h) adsorbents, such as, for example, kaolin andbentonite, and (i) lubricants, such as, for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules, tablets, andpills, the dosage forms may also comprise buffering agents.

Pharmaceutically acceptable adjuvants known in the pharmaceuticalformulation art may also be used in the pharmaceutical compositions ofthe invention. These include, but are not limited to, preserving,wetting, suspending, sweetening, flavoring, perfuming, emulsifying, anddispensing agents. Prevention of the action of microorganisms may beensured by inclusion of various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, and the like. Itmay also be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. If desired, a pharmaceutical compositionof the invention may also contain minor amounts of auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents,antioxidants, and the like, such as, for example, citric acid, sorbitanmonolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.

Solid dosage forms as described above may be prepared with coatings andshells, such as enteric coatings and others well known in the art. Theymay contain pacifying agents, and can also be of such composition thatthey release the active compound or compounds in a certain part of theintestinal tract in a delayed manner, Non-limiting examples of embeddedcompositions that may be used are polymeric substances and waxes. Theactive compounds may also be in microencapsulated form, if appropriate,with one or more of the above-mentioned excipients.

Suspensions, in addition to the active compounds, may contain suspendingagents, such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances, and the like.

Compositions for rectal administrations are, for example, suppositoriesthat may be prepared by mixing at least one crystalline form ofgenistein according to the present disclosure with, for example,suitable non-irritating excipients or carriers such as cocoa butter,polyethyleneglycol or a suppository wax, which may be solid at ordinarytemperatures but may be liquid at body temperature and, therefore, meltwhile in a suitable body cavity and release the active componenttherein.

Because the crystalline form of genistein is maintained duringpreparation, solid dosage forms are preferred for the pharmaceuticalcomposition of the invention. Solid dosage forms for oraladministration, which includes capsules, tablets, pills, powders, andgranules, may be used. In such solid dosage forms, the active compoundmay be mixed with at least one inert, pharmaceutically acceptableexcipient (also known as a pharmaceutically acceptable carrier). Thecrystalline forms of genistein according to the invention may also beused as precursors in the formulation of liquid pharmaceuticalcompositions. Administration of the crystalline forms of genistein inpure form or in an appropriate pharmaceutical composition may be carriedout via any of the accepted modes of administration or agents forserving similar utilities. Thus, administration may be, for example,orally, buccally, nasally, parenterally (intravenous, intramuscular, orsubcutaneous), topically, transdermally, intravaginally, intravesically,intrasystemically, or rectally, in the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, suppositories, pills, soft elastic and hard gelatin capsules,powders, solutions, suspensions, or aerosols, or the like, such as, forexample, in unit dosage forms suitable for simple administration ofprecise dosages. One route of administration may be oral administration,using a convenient daily dosage regimen that can be adjusted accordingto the degree of severity of the disease-state to be treated.

The invention also relates to preparation of a medicament using at leastone crystalline form of genistein for the treatment of a variety ofdiseases. These include, but are not limited to: diseases where theinhibition of one or more protein tyrosine kinase(s) is beneficial, suchas, for example, kinases which are affected by genistein are possibletargets; hyperproliferative diseases such as various cancers, such as,for example, colorectal, breast, lung, prostate, bladder, renal orpancreatic cancer, or leukaemia or lymphoma or proliferativeinflammatory atrophy; diseases where the inhibition of inflammation isbeneficial, such as, for example, chronic inflammation, inflammatorybowel disease, Crohn's disease, Sjögren's disease, rheumatoid arthritis,arthritis, atopic dermatitis, vasculitis, psoriasis, benign prostatehyperplasia, wound healing, end stage renal disease, chronic kidneydisease, chronic obstructive pulmonary disease, asthma; diseases wherethe inhibition of infection is beneficial, such as, for example localinfection, systemic infection, sepsis, systemic fungal infection, localfungal infection; diseases where restoring normal chloride and salt(water) movements in body organs and glands in people is beneficial,such as, for example, stimulating the cystic fibrosis transmembraneconductance regulators as well as diseases and symptoms relating topostmenopausal condition such as hot flushes and osteoporosis as well asdiseases where inhibition of a soluble protein from forming insolubleextracellular fibril deposits causing organ dysfunction is beneficial,such as amyloidosis, for example those where the fibril deposits arecomposed of Transthyretin (TTR), such as Familal Amyloid Polyneuropathy.

EXAMPLES

The following analytical techniques were used in the examples below:

X-Ray Powder Diffraction (XRPD):

X-ray powder diffraction studies were performed on a Bruker D8-Discoverdiffractometer. Approximately 5 mg of sample was gently compressed onthe XRPD zero back ground single 96 well plate sample holder. The samplewas then loaded into a Bruker D8-Discover diffractometer in transmissionmode and analyzed using the experimental conditions shown in Table 1.

TABLE 1 XRPD Measurement Conditions Raw Data Origin BRUKER-binary V3(.RAW) Scan Axis Gonio Start Position [°2Th.] 4.0000 End Position[°2Th.] 49.9800 Step Size [°2Th.] 0.0200 Scan Step Time [s] 39.1393 ScanType Continuous Offset [°2Th.] 0.0000 Divergence Slit Type FixedDivergence Slit Size [°] 2.0000 Specimen Length [mm] 10.00 ReceivingSlit Size [mm] 0.1000 Measurement Temperature [° C.] 25.00 AnodeMaterial Cu K-Alpha1 [Å] 1.54060 K-Alpha2 [Å] 1.54443 K-Beta [Å] 1.39225K-A2/K-A1 Ratio 0.50000 Generator Settings 40 mA, 40 kV DiffractometerNumber 0 Goniometer Radius [mm] 250.00 Dist. Focus-Diverg. Slit [mm]91.00 Incident Beam Monochromator No Spinning No

Differential Scanning Calorimetry (DSC):

Approximately 2 mg of sample was weighed into an aluminum DSC pan andsealed with an aluminum lid (non-hermetically). The sample pan was thenloaded into a Pyris 1 Perkin-Elmer DSC (equipped with a liquid-nitrogencooling unit) cooled and held at 25° C. Once a stable heat-flow responsewas obtained, the sample was then heated to 300° C. at a scan rate of10° C./min and the resulting heat flow response monitored. A 20 cm³/minhelium purge was used to prevent thermally induced oxidation of thesample during heating and also to reduce the thermal lag through thesample to increase the instrument sensitivity. Prior to analysis, theinstrument was temperature and heat-flow calibrated using an indiumreference standard.

Gravimetric Vapor Sorption (GVS):

Approximately 15 mg of sample was placed into a wire-mesh vapor sorptionbalance pan and loaded into an SMS intrinsic vapor sorption balancesupplied (Surface Measurement Systems Instruments). The sample was thendried by maintaining a 0% humidity environment until no further weightchange was recorded. Subsequently, the sample was then subjected to aramping profile from 0-90% RH at 10% RH increments, maintaining thesample at each step until equilibration had been attained (99.5% stepcompletion). Upon reaching equilibration, the % RH within the apparatuswas ramped to the next step and the equilibration procedure repeated.After completion of the sorption cycle, the sample was then dried usingthe same procedure. The weight change during the sorption/desorptioncycles were then monitored, allowing for the hygroscopic nature of thesample to be determined.

Thermogravimetric Gravimetric (TGA):

Approximately 5 mg of sample was accurately weighed into a platinum TGApan and loaded into a Perkin-Elmer TGA 7 gravimetric analyser held atroom temperature. The sample was then heated at a rate of 10° C./minfrom 25° C. to 300° C. during which time the change in weight monitored.The purge gas used was nitrogen at a flow rate of 20 cm³/min. Prior toanalysis, the instrument was weight calibrated using a 100 mg referenceweight and temperature calibrated using an alumel reference standard.

Polarized Light Microscopy (PLM):

The presence of crystallinity (birefringence) was determined using aLeica Leitz DMRB polarized optical microscope equipped with a highresolution Leica camera and image capture software (Firecam V.1.0). Allimages were recorded using 10× objectives unless otherwise stated.

¹H Nuclear Magnetic Resonance (NMR):

¹H NMR was performed on a Bruker AC200 200 MHz Spectrometer. NMR of eachsample was performed in deutero-methanol. Each sample was prepared inca. 5 mg concentration.

Example 1 Crystalline Genistein Sodium Salt Dihydrate

1.1 Preparation of Genistein Sodium Salt Dihydrate:

ca., 300 mg of genistein was placed in 6 cm³ (20 vols) of IPA. Onaddition of 1M sodium hydroxide (NaOH) a reaction was quickly evident(color change from pale yellow to vibrant yellow). The mixture wasallowed to shake at ambient temperature for ca. 3 hrs and then allowedto stand over ca. 2 days (weekend). The solid was isolated by filtrationand allowed to dry at ambient temperature for ca. 24 hrs. The genisteinsodium salt prepared according to this method is crystalline genisteinsodium salt dihydrate which has been characterized by the followingmethods.

1.2 XRPD of Crystalline Genistein Sodium Salt Dihydrate

The XRPD pattern as shown in FIG. 1 was obtained using the proceduredescribed above. As shown in FIG. 1, the XRPD analysis reveals a solidform impurity which is probably an IPA solvate of the sodium salt.Drying the material at 80° C. overnight removes the impurity. The peaksin the XRPD pattern at an experimental °2θ+0.2°2θ are listed in Table 2.The entire list of peaks, or a subset thereof, may be sufficient tocharacterize crystalline genistein sodium salt dihydrate. One subset ofpeaks that, individually or in combination, may be used to characterizecrystalline genistein sodium salt dihydrate from FIG. 1 includes 5.9,11.6, 11.8, 15.2, 24.8, 28.2, 28.9, and 28.9°2θ+0.2°2θ.

TABLE 2 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 5.4 16.5 6.255.9 15.0 14.05 7.5 11.8 21.61 8.2 10.8 4.19 11.6 7.6 6.4 11.8 7.5 7.4214.8 6.0 4.3 15.2 5.8 71.86 16.0 5.5 5.13 16.8 5.3 8.89 17.1 5.2 7.3123.5 3.8 5.6 24.3 3.7 22.41 24.8 3.6 100 25.5 3.5 7.31 26.7 3.3 4.8727.0 3.3 5 28.2 3.2 17.86 28.5 3.1 22.31 28.9 3.1 14.37 29.7 3.0 4.35

1.3 DSC of Dried Crystalline Genistein Sodium Salt Dihydrate

A sample was prepared by drying the crystalline genistein sodiumdihydrate salt, prepared according to the procedure described in 1.1above, at 80° C. overnight. FIG. 2 shows the DSC of the sample of driedcrystalline genistein sodium salt dihydrate. The DSC indicatesdehydration ca. 91° C. followed by melting at ca. 132° C. The otherpeaks are probably associated with degradation (as also indicated by theTGA traces shown in FIGS. 4 and 5, discussed below).

1.4 GVS of Crystalline Genistein Sodium Salt Dihydrate

As shown in FIG. 3, the GVS study of crystalline genistein sodiumdihydrate indicated hydrate formation (GVS cycle dehydrates materialprior to analysis and a maximum of 45 wt % of water adsorbed. However,between 20 and 70 RH % (typical working range of material) only ca. 2%moisture change was observed.

1.5 TGA of Crystalline Genistein Sodium Salt Dihydrate

FIG. 4 shows the TGA trace from a sample of crystalline genistein sodiumsalt dihydrate that was dried at ambient temperature for about 24 hours,1.1 above. FIG. 5 is a TGA from a sample of prepared crystallinegenistein sodium dihydrate salt that was dried at 80° C. overnight. TheTGA indicates that the sodium salt is hydrated and water loss commencesat ca. 75° C., suitable for further development. The weight loss isconsistent with one mole of water to one mole of sodium.

1.6 PLM of Crystalline Genistein Sodium Salt Dihydrate

The PLM of crystalline genistein sodium salt dihydrate showed aneedle-like morphology.

1.7 Solubility Measurements of Crystalline Genistein Sodium SaltDihydrate

Aqueous Solubility: Aqueous solubility was measured using the followingprotocol. Slurries of genistein and of crystalline genistein sodiumdihydrate salt were made up in aqueous media in which pH was set at 4.5,6.7, and 7.5, each slurry was shaken at ambient temperature for ca. 24hours and then filtered using a 0.2 μm filter into a clean vial. Thesaturated solutions were then diluted and analyzed for API (genistein)content using N—Ac-DL-Methionine on a Chirobiotic T HPLC column and UVdetector set at λmax=270 nm. The mobile phase was acetonitrile/water runin isocratic mode over a 30 minute period. The results are presented inTable 3, (BDL=below detection limits). No API peaks were evident fromthe HPLC traces run with genistein (should appear at ca. 6-7 min)indicating that genistein is extremely insoluble in aqueous media andthat the levels are below the sensitivity of the HPLC technique employed(sensitivity of the technique mg to level). Genistein is reported toexhibit an aqueous solubility in the range of 10-40 nM.

TABLE 3 Genistein, Crystalline Genistein Sodium Solvent mg/ml SaltDihydrate, mg/ml Water/slurry pH 4.5 BDL 0.136 Water/slurry pH 6.7 BDL0.707 Water/slurry pH 7.5 BDL 0.650

Solubility in Different Solvents:

Solubility in different organic solvents was measured using thefollowing protocol, Approximately 25 mg portions of genistein andcrystalline genistein sodium dihydrate salt were placed in 48 differentvials, separately, 5 volume aliquots of each solvent were addedexclusively to a vial. Between each addition, the mixture was checkedfor dissolution and if no dissolution was apparent, the procedure wascontinued until dissolution was observed or when 50 volumes had beenadded. The results are shown in Table 4.

TABLE 4 Genistein Crystalline Genistein Solubility, Sodium SaltDihydrate Solvent mg/ml Solubility, mg/ml Methanol (MeOH) <10.6 <8.8Ethanol (EtOH) <10.8 98 2-Propanol (i PA) <10.5 <10.2 Acetone/IPA(50:50) 20.1 20.2 1-Butanol (BuOH) <10.5 19.3 Methyl acetate (MeOAc)<10.3 <10.2 Acetone ca. 17  <10.2 1,4-dioxane <10.3 <10.6 Acetonitrile(MeCN) <10.1 <10.4 Tetrahydrofuran (THF) 26.3 <10.4 Dichloromethane(DCM) <10.3 <10.8 tert-Butylmethyl ether (TBME) <10.5 <9.2 Methylethylketone (MEK) <10.8 <9.2 Heptane <10.5 <9.9 Octanol <10.5 <11N-N-dimethylformamide (DMF) ca. 100 >217.6 Dimethyl sulfoxide (DMSO) ca.100 >235.2 Toluene <10.1 <9.9 N-Methyl-2-pyrrolidinone (NMP) ca. 68 90.4 Methyl isobutyl ketone (MIBK) <10.1 <9.0 Acetone/Water (50:50)<10.1 69.6 Toluene/Dioxane (50:50) <10.1 <9.7 Cyclohexane <10.7 <10.9Diisopropylether (DIPE) <10.6 <9.7

1.8 Stability Study of Crystalline Genistein Sodium Salt Dihydrate

Sample stability was tested at 80° C. for 7 days and at 40° C./75 RH %for 7 days. Observations such as color change were noted after 7 daysand XRPD of samples were taken after 7 days to investigate any solidform change. FIG. 6 shows the XRPD patterns of the original sample andsamples of crystalline genistein sodium salt dihydrate at 80° C. for 7days and at 40° C./75 RH % for 7 days. The 40° C./75 RH % studyindicated no change over a 7 day period. Storing the material at 80° C.over a 7 day period indicated a slight loss in crystallinity suggestingslow dehydration. The 7 day light stability tests revealed no change incolor or solid form.

1.9 ¹H NMR Spectrum of Crystalline Genistein Sodium Salt Dihydrate

FIG. 7 illustrates the ¹H NMR spectrum of the crystalline genisteinsodium salt dihydrate. Table 5 lists the peaks in the ¹H NMR spectrum.Displacement of the chemical shifts for the aromatic protons at ca. 5.9in genistein to 6.1 ppm in the ¹H NMR of FIG. 8 confirms salt formation.

TABLE 5 Chemical Shift Mulitlpicity Range (ppm) 7.952 s 7.932-7.9197.372 m 7.429-7.306 6.861 m 6.927-6.791 6.101 dd 6.187-6.028 4.936 s5.256-4.723 3.34 q 3.577-3.096 1.085 s 1.213-0.934 s = singlet, m =multiplet, dd = doublet of doublet, q = quadruplet

1.10 Disproportionation Study of Crystalline Genistein Sodium SaltDihydrate

A 50 mg sample of crystalline genistein sodium salt dihydrate wasslurried in 250 μl distilled water for ca, 48 hours and then checked byXRPD for disproportionation. The pH of the supernatant was also measuredusing a Corning 240 pH meter. No signs of disproportionation wereobserved. The pH of the supernatant after slurrying was 7.1.

1.11 Hydration Study of Crystalline Genistein Sodium Salt Dihydrate

Approximately 100 mg of crystalline genistein sodium salt dihydrate wereplaced in ca. 500 μL IPA/water mixtures (3%, 5% and 10%) at the waterlevel. Each mixture was agitated for ca. 48 hours at ambient temperatureand then filtered to recover the solid for XRPD and TGA studies. Asshown in FIG. 8, hydration was indicated from a change from the originalmaterial in the XRPD pattern corresponding with weight loss from TGA(material depending). The hydration study revealed no further hydrates;but removed the IPA solvate impurity.

1.12 Single Crystal X-Ray Diffraction of Crystalline Genistein SodiumSalt Dihydrate

Single Crystal Preparation:

Crystals were grown from solutions of crystalline genistein sodium saltdihydrate (ca. 48 mg) dissolved in 50:50 IPA/Water (3 cm³). The solutionwas then allowed to slowly evaporate through pierced parafilm,Needle-like crystals were apparent after ca. 2 weeks of evaporation.

Single Crystal X-Ray Diffraction:

A lath-like needle of the sample was selected for data collection.Diffraction data were collected with Mo—Kα radiation using a BrukerSmart Apex CCD diffractometer equipped with an Oxford Cryosystemslow-temperature device operating at 150 K.

On indexing the data set, the crystal structure was determined to bepseudosymmetric. Strong data could be indexed on a primitive.,metrically monoclinic, cell of dimensions a=3.76, b=30.23, c=12.12 Å,β=106.2°, V=1324 Å³. A complete indexing of all data could only beobtained with a larger triclinic cell of dimensions a=7.52, b=11.65,c=30.46 Å, α=89.8°, β=82.9°, γ=88.1°, V=2647 Å³. This cell is itselftransformable to a pseudo monoclinic C-centred cell of dimensionsa=7.52, b=60.46, c=11.65 Å, β=91.9°, V 5295 Å³.

The diffraction data were integrated and reduced (SAINT), and correctedfor systematic errors using the multiscan procedure SADABS. Thestructure was solved in P-1 by direct methods (SHELXS) using thedata-set integrated on the triclinic cell described above. The structurewas refined against |F|² using all data (SHELXL). Incorporation of atwin law was necessary for completion of the structure. The twin lawused was a two-fold rotation about the [−1 0 2] direction, whichcorresponds to the b-axis direction of the monoclinic cells describedabove.

In addition to being twinned, the structure is pseudosymmetric. Thismeans that the atomic coordinates within the organic fragments arerelated to each other, and it results in correlations and mathematicalinstabilities into the least squares refinement. In order to overcomethese similarities, restraints were applied to all chemically relatedbond distances and angles. Pairs of molecules (1 and 2, and 3 and 4) arerelated by a translation of a/2, and so equivalent anisotropicdisplacement parameters were constrained to be equal. Some damping wasneeded to achieve convergence. Correlation also causes equivalent bondlengths to become artificially different, and care should be taken notto ascribe any significance to apparent differences in chemicallyequivalent bond distances, for example. A more elaborate refinementmodel would be needed to resolve these effects.

Hydrogen atoms attached to carbon were placed in calculated positions.Some hydrogen atoms attached to oxygen could be located in differencemaps. In particular, the H-atoms were attached to the O-atoms ligatingthe sodium ions (O141 and O144). Positions for ligand-water H-atoms werelocated in a Fourier map calculated about the loci of possibleH-positions; those making geometrically sensible H-bonds and avoidingshort contacts were included in the model, H-atoms attached to O8 werelocated in a difference map, and the whole molecule then initiallyrefined as a rotating rigid group, thereafter the H-atoms were treatedwith a riding model. The remaining H-atoms (H7A and H142) were placedalong short O . . . O vectors. There was no evidence in Fourier maps forH-atoms on O42 and O43, and attempts to place them led to thedevelopment of unreasonably short H . . . H contacts with other H-atoms.

The final ‘conventional’ R-factor [based on F and 7355 data withF>4σ(F)] was 0.0616. Other crystal and refinement parameters are listedin Table 6.

TABLE 6 Single Crystal Data and Structure Refinement for the CrystallineGenistein Sodium Salt Dihydrate. A. CRYSTAL DATA Empirical formulaC₆₀H₅₄Na₂O₂₈, C₃₀H₃₂Na₂O₁₆, 2(C₁₅H₉O₅), 2(H₂O) Formula weight 1269.01Wavelength 0.71073 A Temperature 150(2) K Crystal system Triclinic Spacegroup P-1 Unit cell dimensions a = 7.524(2) A alpha = 89.762(7) deg. b =11.646(3) A beta = 82.902(10) deg. c = 30.464(6) A gamma = 88.073(10)deg. Volume 2647.4(12) A{circumflex over ( )}3 No. of reflections forcell 7324 (2.5 < theta < 25 deg.) Z 2 Density (calculated) 1.592Mg/m{circumflex over ( )}3 Absorption coefficient 0.141 mm{circumflexover ( )}−1 F(000) 1320 B. DATA COLLECTION Crystal description colorlessneedle-like lath Crystal size 0.70 × 0.16 × 0.10 mm Instrument BrukerSmart Apex CCD Theta range for data collection 0.67 to 24.55 deg. Indexranges −8 <= h <= 8, −13 <= k <= 13, −35 <= l <= 35 Reflectionscollected 33227 Independent reflections 8758 [R(int) = 0.0574] Scan typeomega Absorption correction Multiscan, (Tmin = 0.804, Tmax = 0.984) C.SOLUTION AND REFINEMENT Solution direct (SHELXS-97 (Sheldrick, 2008))Refinement type Full-matrix least-squares on F{circumflex over ( )}2Program used for refinement SHELXL-97 Hydrogen atom placementgeom/difmap Hydrogen atom treatment riding/rotating groupData/restraints/parameters 8758/1636/580 Goodness-of-fit on F{circumflexover ( )}2 1.088 Conventional R [F > 4sigma(F)] R1 = 0.0616 [7355 data]Weighted R (F{circumflex over ( )}2 and all data) wR2 = 0.1489 Finalmaximum delta/sigma 0.073 Weighting scheme calc w = 1/[\s{circumflexover ( )}2{circumflex over ( )}(Fo{circumflex over ( )}2{circumflex over( )}) + (0.0598P){circumflex over ( )}2{circumflex over ( )} + 2.2931P]where P = (Fo{circumflex over ( )}2{circumflex over ( )} +2Fc{circumflex over ( )}2{circumflex over ( )})/3 Largest diff. peak andhole 0.316 and −0.310 e.A{circumflex over ( )}−3

Discussion:

The single crystal structure of crystalline genistein sodium saltdihydrate shows that the compound has an overall formula of[Na₂(H₂O)₄(μ-H₂O)₂(LH)₂]L₂.2H₂O where LH=the fully protonated genisteinligand C₁₅H₁₀O₅ and μ-H₂O are bridging water molecules between the Naions (i.e., the Na ions are each bonded to two terminal waters and twobridging waters (designated μ-H₂O), plus one LH ligand—see FIG. 9). Thisconclusion depends on the model of H-atom placement described above.Hydrogen atom placement using X-ray data is usually regarded astentative, the more so here because of the problems encountered duringstructure analysis. That being said, the H-atom positions proposed doform a plausible H-bonding set with all H-atoms involved ingeometrically normal hydrogen bonds.

As shown in FIG. 9, the cationic sodium complexes consist of dimericunits formed across inversion centres. The sodium ions arefive-coordinate, the coordination sphere consisting of two terminal andtwo bridging water ligands and one of the LH ligands. A hydrogen bond isformed between the ligating alcohol moiety and one of the terminal watermolecules (H141 . . . O1 and H141 . . . O4). The L⁻ anions aredeprotonated at the phenolic O42 and O43 sites. The C—O⁻ distances arequite short/average 1.34 Å). An internal hydrogen bond is formed betweenH6* and O8* the ligating LH and the L⁻ anions.

FIG. 9 illustrates the centrosymmetric disodium cation in the dimericstructure of crystalline genistein sodium salt dihydrate, wherein theintramolecular hydrogen bonds are shown as dashed lines.

The packing in the crystal is dominated by hydrogen bonding. The cationsare linked to the anions via water molecules to form layers which alsofeature stacking interactions between cations and anions. FIG. 10 showsone such layer involving cations based on O11 and anions based on O12.Water molecules are shown, in turquoise. The view is along [010].

Similar layers composed of molecules based on O13 and O14 are alsoformed, and the two types of layers alternate along the b-axis, beinglinked by H-bonds. FIG. 11 illustrates the overall picture as a threedimensional network. FIG. 11 illustrates the packing of crystallinegenistein sodium salt dihydrate viewed along the [100] direction.

Analysis using the PLATON/MISSYM procedure indicates that the organicfragments on their own can be described using the small (1324 Å³ celland space group P2₁/c, and it is only the sodium ions and watermolecules which break this symmetry, explaining the pattern of strongand weak data in the diffraction pattern, and the pseudosymmetryproblems experienced in refinement.

The calculated XRPD pattern based on the single crystal data andstructure for the crystalline genistein sodium salt dihydrate is shownin FIG. 12. Table 7 lists the peaks in the calculated XRPD pattern. Theentire list of peaks, or a subset thereof, may be sufficient tocharacterize crystalline genistein sodium salt dihydrate. One subset ofpeaks that, individually or in combination, may be used to characterizecrystalline genistein sodium salt dihydrate from FIG. 12 includes 5.8,11.6, 15.2, 17.6, 25.1, 28.4, 28.8, and 29.2°2θ±0.2°2θ.

TABLE 7 d-spacing Rel. Int. Pos. [°2θ] [Å] [%] 5.8 15.1 23.520 8.1 10.95.770 11.6 7.6 12.620 15.2 5.8 69.700 17.6 5.0 5.570 23.7 3.8 4.540 24.63.6 35.590 25.0 3.6 32.830 25.1 3.6 97.090 25.1 3.6 98.070 25.1 3.5100.000 25.2 3.5 73.730 25.3 3.5 14.020 25.3 3.5 9.220 25.7 3.5 7.90025.8 3.5 10.480 28.4 3.1 23.020 28.5 3.1 15.880 28.8 3.1 10.520 28.8 3.127.560 29.2 3.1 23.560 29.2 3.1 22.670 32.3 2.8 7.540

1.13 Bioavailability of Genistein Alone and From Crystalline GenisteinSodium Salt Dihydrate, Following Intraduodenal and IntravenousAdministration in Male Sprague-Dawley Rats.

Preparation of Dosing Solutions for In-Vivo Study:

Genistein and Crystalline Genistein sodium salt dihydrate were stored atroom temperature under desiccant and protected from light. The dosingsolutions were prepared fresh from powders on the day of dosing. Thedosing solution for intravenous administration (IV) was prepared at 1mg/mL (free acid) in 50:50 DMSO:saline. The dosing solutions forintraduodenal administration (ID) were prepared at 2 mg/mL (genisteinfree acid) in a 0.2% sodium carboxymethyl cellulose (Na CMC) solution inwater.

Animal Dosing:

The pharmacokinetics of genistein was evaluated in fasted maleSprague-Dawley rats. Each animal was fitted with a jugular vein cannula(JVC) for blood sampling. Animals intended for intravenous dosing werefitted with an additional JVC for dose administration, Animals intendedfor intraduodenal dosing were fitted with an intraduodenal cannula (IDC)for dose administration. Surgically modified animals were housed one percage. All animals were supplied with a commercial rodent diet (LabDiet,Certified Rodent Diet #5002) ad libitum prior to study initiation. Foodwas then withheld from the animals for a minimum of twelve hours beforethe study and during the study, until eight hours post dose when foodwas returned. Water was supplied ad libitum.

Intraduodenal dosing solutions were administered as a single bolus doseat time zero on the day of dosing. Intravenous doses were administeredas a slow IV injection over approximately 1 minute. Blood sampling timesbegan at the end of the infusion. Blood samples were collected. Thestudy design is shown in Table 8.

TABLE 8 Out-line of Comparative Pharmacokinetic Study of Genistein andCrystalline Genistein Sodium Salt Dihydrate, in Rats. Dosing SolutionDosing Blood Treatment Test Dosing Dose Conc Volume Sampling GroupCompound Route (mg/kg) (mg/ml) (ml/kg) Vehicle Time points 1 GenisteinID 20 10 2 0.2% Pre dose, 15, NaCMC 30 min, 1, 2, 3, In water 4, 6, 8and 24 h 2 Crystalline ID 20 10 2 0.2% Pre dose, 15, Genistein NaCMC 30min, 1, 2, 3, Sodium In water 4, 6, 8 and 24 h Salt Dihydrate 3Genistein IV 1 1 1 50% Pre dose, 2, 5, DMSO in 15, 30 min, 1, saline 2,3, 4, 8 and 24 h 4 Crystalline IV 1 1 1 50% Pre dose, 2, 5 GenisteinDMSO in 15, 30 min, 1, Sodium saline 2, 3, 4, 8 and Salt 24 h Dihydrate

Each blood sample was collected from the rats via a jugular vein cannulaand placed into chilled polypropylene tubes containing sodium heparin asan anticoagulant. Samples were centrifuged at a temperature of 4″C andat a speed of 13,000 rpm for 5 minutes, Samples were maintained chilledthroughout processing. Each plasma sample was split into two aliquots.The first aliquot contained 50 μl of plasma. All remaining plasma volumewas used for the second aliquot. Samples were then placed on dry ice,and stored in a freezer set to maintain −60° C. to −80° C. The totalconcentration of genistein in plasma samples were analyzed by LC-MS/MSafter an overnight incubation with glucuronidase/arylsulfatase enzymemixture. Pharmacokinetic parameters were calculated using the WinNonlinsoftware.

Analysis of Plasma Samples:

An LC-MS/MS analytical method for the determination of genistein in ratplasma was developed. Prior to sample analysis, a standard curve wasanalyzed to determine the specificity, range, and linearity of themethod. Total genistein in plasma samples was determined by pre-treatingall samples with β-glucuronidase/arylsulfatase enzymes and incubatingprior to analysis. Incubation with the enzyme mix deconjugated anyglucuronide or sulfate metabolites of genistein back to the parent form.

Acceptance Criteria for LC-MS/MS Analysis:

One standard curve was dispersed throughout each analytical run. Atleast ⅝ of the standards must be accurate to within ±20%, except at theLLOQ where ±25% is acceptable, in order for the run to pass.

Pharmacokinetic Analysis:

Individual plasma concentrations versus time data for genistein weresubjected to non-compartmental analysis using the pharmacokineticprogram WinNonlin v. 4.1. Plasma concentrations below the limit ofquantitation (10 ng/mL) were assigned a value of zero for PK analysisonly.

Results:

As shown in FIG. 13, the mean plasma concentration and PK profiles ofgenistein compared with crystalline genistein sodium salt dihydrate wasmarkedly different, following ID dosing. The mean peak plasmaconcentration (C_(max)) of genistein from crystalline genistein sodiumdihydrate salt was 4.2 fold higher compared to the peak plasmaconcentration of genistein, 8330±2176 ng/mL and 1983±1130 ng/mL,respectively. Already within 15 minutes after ID dosing of crystallinegenistein sodium salt dihydrate maximum plasma concentration (C_(max))of genistein was observed, while the C_(max) of genistein was observedat 2 hours post dose (FIG. 13 and Table 10). The genisteinbioavailability from crystalline genistein sodium salt dihydrate was55±16% compared to 16±4.4% for genistein (Table 9).

TABLE 9 Pharmacokinetic Parameters after Intraduodenal Administration of20 mg/kg of Respective Form (mean ± SD, n = 3). Crystalline Genistein PKparameter Genistein Sodium Salt Dihydrate C_(max) (ng/ml) 1983 ± 11308330 ± 2176 t_(max) (H) 2.0 ± 0   0.83 ± 1.0  AUC_(last) (h · kg ·ng/ml/mg) 414 ± 111 1161 ± 358  Bioavailability (%)  16 ± 4.4 55 ± 16

As shown in Table 10, the pharmacokinetic profile of genistein andcrystalline genistein sodium salt dihydrate following IV dosing were notsignificantly different between the two forms.

TABLE 10 Pharmacokinetic Parameters after Intravenous Administration of1 mg/kg of Respective Form (mean ± SD, n = 3). Crystalline Genistein PKparameter Genistein Sodium Salt Dihydrate C₀ (ng/ml)¹ 6617 ± 1059 6640 ±1223 T_(1/2) (h) 1.4 ± 0.3 1.6 ± 0.9 CL (L/h/kg) 0.40 ± 0.09 0.47 ± 0.08Vss (L/kg) 0.40 ± 0.06 0.36 ± 0.09 AUC_(last) (h · kg · ng/ml/mg) 2533 ±638  2129 ± 331  AUC_(∞) (h · kg · ng/ml/mg) 2584 ± 639  2189 ± 356 ¹extrapolated to t = 0.

1.14 Physicochemical Characterization and the Kinetic and EquilibriumSolubility Comparison Between Genistein and Crystalline Genistein SodiumSalt Dihydrate.

Crystalline genistein sodium salt dihydrate shows superior early andlate intrinsic kinetic solubility profiles as compared to genistein inEtOH/dH₂O solutions. The low late intrinsic kinetic solubility ofcrystalline genistein sodium salt dihydrate in 100% EtOH has lesspractical implications for the preclinical development given thenon-physiological nature of the solvent.

Experimental

Genistein and crystalline genistein sodium salt dihydrate were run in aSuperSol 1000 (PREVENTOR Gmbh) solubility assay and the concentration ofthe compounds measured over time in a closed system by measuring theabsorbance in a flow-through measuring chamber at a wavelength of 250nm. Since both compounds form suspensions in pure deionized H₂O,physicochemical properties were assessed from solutions of 100% EtOH aswell as mixtures of dH₂O and EtOH, specifically, EtOH 50/50 (vol/vol)and EtOH 75/25 (vol/vol) according to European Pharmacopeia guidelinesJanuary 2008, Section 2.9.3., Table 2.9.3.5.

The following parameters were measured:

t_([MSS]) defined as: Time from start of analysis to MaximumSolubilization Speed (min)

C_([MSS]) defined as: Early kinetic solubility as expressed asconcentration at Maximum Solubilization Speed (mg×l⁻¹)

C_([Eq]) defined as: Late kinetic solubility as expressed asconcentration at Equilibrium Kinetic Solubility (mg×l⁻¹)

Δt_([Eq]) defined as: Time from start of analysis to Equilibrium Kineticsolubility (min)

ΔC[C_(Eq)-C_(MSS)] defined as: Difference in concentration between Earlyand Late Kinetic Solubility as defined above (mg×l⁻¹)

Δt[C_(Eq)-C_(MSS)] defined as: Difference in time between Early and LateKinetic Solubility endpoints (min)

MSS defined as: Maximum Solubility Speed defined by C_([MSS])/t_([MSS])(mg×l⁻¹×min⁻¹)

ISI defined as: Intrinsic Solubility Index defined by ΔC[C_(Eq)-C_(MSS)]/Δt [C_(Eq)-C_(MSS)]

The higher the ISI value, the faster the solubilization and the strongerthe relative contribution of the late, intrinsic kinetic equilibriumsolubility C_([eq]).

KSR defined as: Kinetic Solubility ratio given by C_([MSS])/C_([Eq]).

The KSR is the numerical ratio indicator of the relative contribution ofthe early kinetic Solubility to the Overall Late Kinetic EquilibriumSolubility. The higher the KSR Value, the stronger the relativecontribution of the early kinetic solubility C_([MSS]).

Results: The thermodynamic kinetic and equilibrium solubility data ofgenistein and crystalline genistein sodium salt dihydrate were assessedunder the conditions reported in Tables 11, 12, and 13.

As shown in Table 11, genistein showed (a) good MSS, (b) acceptable KSRand (c) good late solubility profiles, while crystalline genisteinsodium salt dihydrate showed (a) excellent MSS (b) excellent KSR and (c)good-to-acceptable ISI. For EtOH/dH₂O 50/50 (vol/vol) crystallinegenistein sodium salt dihydrate displayed the best early intrinsickinetic solubility profile.

TABLE 11 EtOH/dH₂O 50/50 (vol/vol) ΔC Δt [C_(Eq) − [C_(Eq) − MSSt_([MSS]) C_([MSS]) C_([Eq]) t_([Eq]) C_(MSS)] C_(MSS)] ISI KSRGenistein 12.18 0:61 7.43 12.87 4:12 5.44 3:51 1.55 0.58 Crystalline20.66 0:71 14.67 17.40 5:28 2.73 4:57 0.60 0.84 Genistein Sodium SaltDihydrate

For EtOH/dH₂O 75/25 (vol/vol), as shown in Table 13, genistein showed(a) good MSS, (b) good KSR and (c) good late solubility profiles.Crystalline genistein sodium salt dihydrate showed (a) excellent MSS (b)excellent KSR and (c) excellent ISI, which is the best early and lateintrinsic kinetic solubility profile.

TABLE 12 EtOH/dH₂O 75/25 (vol/vol) ΔC Δt [C_(Eq) − [C_(Eq) − MSSt_([MSS]) C_([MSS]) C_([Eq]) t_([Eq]) C_(MSS)] C_(MS)] ISI KSR Genistein19.67 0:44 8.52 14.03 4:15 5.51 3:71 1.49 0.61 Crystalline 37.33 0:3011.20 15.86 4:01 4.66 3:71 1.26 0.71 Genistein Sodium Salt Dihydrate

As reported in Table 13, at EtOH 100%, genistein showed (a) good MSS,(b) acceptable KSR and (c) good late solubility profiles, whilecrystalline genistein sodium salt dihydrate, in comparison, showed (a)excellent MSS (b) excellent KSR and (c) poor ISI. Crystalline genisteinsodium salt dihydrate showed the best early intrinsic kinetic solubilityprofile, but small contribution to the overall profile.

TABLE 13 EtOH 100% ΔC Δt MSS t_([MSS]) C_([MSS]) C_([Eq]) t_([Eq]) [C, −C_(MSS)] [C_(Eq) − C_(MS)] ISI KSR Genistein 23.81 0:24 5.81 13.79 4:037.98 3:79 2.11 0.42 Crystalline 36.90 0:29 10.85 11.20 5:14 0.35 4:850.07 0.97 Genistein Sodium Salt Dihydrate

1.15 Large Scale Synthesis of Crystalline Genistein Sodium SaltDihydrate

Synthesis:

Crystalline genistein sodium salt dihydrate was prepared on a kilogramscale using the following procedure:

-   -   1. 5.2 kg of 2-propanol (IPA) and 320 g of neutral genistein        were charged into a 15 L glass reactor.    -   2. The temperature of the mixture was adjusted to 22±3° C. and        632 g of 2M aq. NaOH was added dropwise during about 40 minutes        at 22±4° C.    -   3. The mixture was agitated at 22±4° C. for about 19 hours and        cooled to about 15° C. and agitated for 4 hours.    -   4. The mixture was agitated at temperature cycles (15±3°        C.→35±3° C. during 1 h→35±3° C. for 4 h→15±3° C. during 1        h→15±3° C. for 4 h) for about 90 hours and finally at 15±3° C.        for about 4.5 h.    -   5. The precipitated product was filtered and washed with 1.2 kg        of pre-cooled 2-propanol.    -   6. The filtered product was dried in vacuum tray dryer without        vacuum at first at the set temperature of 30° C. for about 19 h,        then at the set temperature of 40° C. for about 20 h, then at        the set temperature of 50° C. for about 24 h, then at the set        temperature of 60° C. for about 16 h and finally at set        temperature of 70° C. for about 10 h until the water content        measured by KF-titration met set specification.    -   7. Finally, the product (0.24 kg) was ground and packed into        PE-bags.

Optional Recrystallization Procedure

Crystalline genistein sodium salt dihydrate was recrystallized using thefollowing procedure:

-   -   1. 24 g of the crystalline genistein sodium salt dihydrate        prepared as above was added to 240 ml of ethanol.    -   2. This mixture was stirred at 250 rpm and heat at 45° C., for        ca. 30 mins.    -   3. The resulting solution was allowed to cool to room        temperature.    -   4. Heptane was then added in aliquots (detailed as follows)        adding 1 aliquot per 1 min. Intermittent stirring at 40 rpm was        used between each addition.        -   Added 4.151 ml of heptane and stirred intermittently at 40            rpm.        -   Added 3.272 ml of heptane and stirred intermittently at 40            rpm.        -   Added 5.209 ml of heptane and stirred intermittently at 40            rpm.        -   Added 3.505 ml of heptane and stirred intermittently at 40            rpm.        -   Added 3.885 ml of heptane and stirred intermittently at 40            rpm.        -   Added 5.465 ml of heptane and stirred intermittently at 40            rpm.        -   Added 6.314 ml of heptane and stirred intermittently at 40            rpm.        -   Added 6.656 ml of heptane and stirred intermittently at 40            rpm.        -   Added 8.258 ml of heptane and stirred intermittently at 40            rpm.        -   Added 6.969 ml of heptane and stirred intermittently at 40            rpm.        -   Added 11.115 ml of heptane and stirred intermittently at 40            rpm.        -   Added 10.750 ml of heptane and stirred intermittently at 40            rpm.        -   Added 14.219 ml of heptane and stirred intermittently at 40            rpm.        -   Added 9.261 ml of heptane and stirred intermittently at 40            rpm.        -   Added 14.913 ml of heptane and stirred intermittently at 40            rpm.        -   Added 13.471 ml of heptane and stirred intermittently at 40            rpm.        -   Added 15.753 ml of heptane and stirred intermittently at 40            rpm.        -   Added 19.172 ml of heptane and stirred intermittently at 40            rpm.        -   Added 23.441 ml of heptane and stirred intermittently at 40            rpm.        -   Added 25.503 ml of heptane and stirred intermittently at 40            rpm.        -   Added 26.856 ml of heptane and stirred intermittently at 40            rpm.        -   Added 28.126 ml of heptane and stirred intermittently at 40            rpm.        -   Added 28.070 ml of heptane and stirred intermittently at 40            rpm.        -   Added 36.738 ml of heptane and stirred intermittently at 40            rpm.        -   Added 35.989 ml of heptane and stirred intermittently at 40            rpm.        -   Added 49.677 ml of heptane and stirred intermittently at 40            rpm.        -   Added 50.145 ml of heptane and stirred intermittently at 40            rpm.        -   Added 32.579 ml of heptane and stirred intermittently at 40            rpm.        -   Added 61.538 ml of heptane and stirred intermittently at 40            rpm.        -   Added 57.143 ml of heptane and stirred intermittently at 40            rpm.        -   Added 51.948 ml of heptane and stirred intermittently at 40            rpm.        -   Added 90.909 ml of heptane and stirred intermittently at 40            rpm.    -   5. The sample was then left to crystallise overnight at room        temperature (ca. 18 hrs).    -   6. The crystalline product was collected by vacuum filtration,    -   7. The crystalline product was then dried for ca. 21 hours while        monitoring the water content by Karl Fischer titration to avoid        the risk of dehydration.

FIG. 14 shows the XRPD pattern of the recrystallized crystallinegenistein sodium salt dihydrate. The peaks in the XRPD pattern at anexperimental °2θ±0.2°2θ are listed in Table 14. The entire list ofpeaks, or a subset thereof, may be sufficient to characterizecrystalline genistein sodium salt dihydrate. One subset of peaks that,individually or in combination, may be used to characterize crystallinegenistein sodium salt dihydrate from FIG. 14 includes 6.0, 7.1, 11.8,11.9, 15.3, 17.8, 21.3, 25.0, 28.3, 28.6, and 29.1°2θ±0.2°2θ. Preferredsubset of peaks include 6.0, 7.1, 15.3, 25.0 and at least two of thethree peaks 28.3, 28.6, and 29.1°2θ±0.2°2θ and 6.0, 7.1, 15.3, 25.0 and28.3°2θ±0.2°2θ.

TABLE 14 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 6.0 14.6 33.537.1 12.4 21.77 8.3 10.7 5.53 11.8 7.5 15.05 11.9 7.4 12.04 15.3 5.890.11 15.6 5.7 5.91 17.8 5.0 17.38 19.3 4.6 7.99 21.3 4.2 10.78 22.1 4.04.67 23.4 3.8 5.61 23.7 3.8 9.49 24.5 3.6 35.37 25.0 3.6 100 25.4 3.59.09 25.6 3.5 16.02 25.8 3.5 18.24 27.6 3.2 6.76 28.3 3.2 21.60 28.6 3.122.15 29.1 3.1 20.45 29.9 3.0 5.12 30.7 2.9 12.80 30.8 2.9 9.80 32.1 2.85.32 35.4 2.5 8.63 35.7 2.5 8.78 36.6 2.5 5.38 39.1 2.3 5.66 40.5 2.26.66 41.0 2.2 8.72 41.1 2.2 7.29 41.9 2.2 8.62 42.6 2.1 8.97

Example 2 Crystalline Genistein Potassium Salt Dihydrate

2.1 Preparation of Genistein Potassium Salt:

ca., 300 mg of genistein was placed in 6 cm³ (20 vols) of IPA, Onaddition of 1M potassium hydroxide (KOH) a reaction of the slurry wasevident (i.e., slurry to clear solution). The mixture was allowed toshake at ambient temperature for ca. 3 hrs during which timeprecipitation was evident. The mixture was then allowed to stand atambient temperature for ca. 2 days (weekend). The solid was isolated byfiltration and allowed to dry at ambient temperature for ca. 24 hrs.

Crystalline genistein potassium salt dihydrate forms from the amorphouspotassium salt upon standing when open to air under ambient roomconditions. It may also be prepared from the amorphous potassium saltwhen the genistein potassium salt is slurried in IPA/water mixture asdescribed in the hydration studies to form crystalline genisteinpotassium salt dihydrate by water uptake.

Genistein potassium salt, thus, appears to be an unstable anhydrousamorphous salt at the point of recovery, which then rapidly absorbswater from the surroundings to crystallize into a dihydrated material.This finding is supported by the light stability test; the hydrationstudy; the 40° C./75 RH % study; the 80° C. storage study and the TGAtest—all described below. The 80° C. storage study is particularlynotable as the material appears to be absorbing water at this elevatedtemperature; thus, suggesting that the hydrate is stable at 80° C. TheGVS data also indicates that the genistein potassium salt dihydrate isthe most stable version and therefore developable. Although thesynthetic procedure described above does not yield the dihydratematerial directly, it may well be produced by further processing orchanging the solvent system to include a higher water content, (i.e., 3%water/IPA), Like the crystalline genistein sodium salt dihydrate, therisk of dehydration in milling is somewhat mitigated by the 80° C.storage tests.

2.2 XRPD of the Amorphous Genistein Potassium Salt

As shown in FIG. 15, the XRPD analysis reveals the solid genisteinpotassium salt produced as described in 2.1 is amorphous (i.e., nopeaks).

2.3 XRPD of the Crystalline Genistein Potassium Salt Dihydrate

FIG. 16 shows the XRPD pattern of the crystalline genistein potassiumsalt dihydrate. The peaks in the XRPD pattern at an experimental°2θ±0.2°2θ are listed in Table 15. The entire list of peaks, or a subsetthereof, may be sufficient to characterize crystalline genisteinpotassium salt dihydrate. One subset of peaks that, individually or incombination, may be used to characterize crystalline genistein potassiumsalt dihydrate from FIG. 16 includes 11.6, 14.5, 14.8, 24.5, 25.2, 27.6,28.0, and 28.4°2θ±0.2°2θ. A preferred subset of peaks includes 11.6,14.5, 24.5, 25.2 and at least two of the three peaks 27.6, 28.0, and28.4°2θ±0.2°2θ.

TABLE 15 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 11.6 7.6 12.9514.5 6.1 51.58 14.8 6.0 30.96 18.0 4.9 4.42 22.3 4.0 4.25 22.6 3.9 11.4724.0 3.7 15.84 24.2 3.7 20.16 24.5 3.6 100 25.2 3.5 19.35 25.6 3.5 8.2827.0 3.3 11.63 27.1 3.3 12.14 27.6 3.2 31.86 28.0 3.2 29 28.4 3.1 23.2129.3 3.0 4.53 29.5 3.0 4.67 30.0 3.0 5.34 30.5 2.9 5.25 30.9 2.9 4.4631.3 2.9 6.03 31.7 2.8 5.5 35.0 2.6 4.76

2.4 PLM of Crystalline Genistein Potassium Salt Dihydrate

PLM analysis of genistein potassium salt dihydrate showed that thepotassium salt is crystalline and has a needle-like morphology. Theneedles are thicker than those of the crystalline genistein sodium saltdihydrate.

2.5 TGA of Crystalline Genistein Potassium Salt Dihydrate

As shown in FIG. 17, TGA indicates that the potassium salt is hydratedand water loss commences at ca. 75° C., suitable for furtherdevelopment. The weight loss is consistent with 2 moles of water to 1mole of potassium.

2.6 DSC of Crystalline Genistein Potassium Salt Dihydrate

As shown in FIG. 18, DSC indicates dehydration ^(c)a. 91° C. with nomelting. The other peaks are probably associated with degradation (asalso indicated by the TGA, FIG. 16).

2.7 GVS of Crystalline Genistein Potassium Salt Dihydrate

As shown in FIG. 19, GVS studies indicate hydrate formation (GVS cycledehydrates material prior to analysis and a maximum of 16 wt % of wateradsorbed. However, between 20 and 70 RH % (typical working range ofmaterial) only ca. 3% moisture change is observed. This is a valuableproperty for pharmaceutical development.

2.8 Solubility Study of Crystalline Genistein Potassium Salt Dihydrate

The aqueous solubility of crystalline genistein potassium salt dihydratewas measured using the protocol described in Example 1.7. Table 16compares the aqueous solubilities of crystalline genistein potassiumsalt dihydrate with that of genistein.

TABLE 16 Crystalline Genistein Genistein, Potassium Salt Dihydrate,Solvent mg/ml mg/ml Water/slurry pH 4.5 BDL 0.04 Water/slurry pH 6.7 BDL0.057 Water/slurry pH 7.5 BDL 0.069

2.9 ¹H NMR of Crystalline Genistein Potassium Salt Dihydrate

FIG. 20 illustrates the ¹H NMR spectrum of the crystalline genisteinpotassium salt dihydrate. Table 17 lists the peaks in the ¹H NMRspectrum. Displacement of the chemical shifts for the aromatic protonsat ca. 5.9 in genistein to 6.1 ppm in the ¹H NMR of FIG. 20 confirmssalt formation.

TABLE 17 Chemical Shift Mulitlpicity Range 7.959 S 8.009-7.941 7.375 m7.437-7.322 6.861 m 6.932-6.800 6.154 dd 6.246-6.068 4.949 s 5.148-4.7143.34 q 3.444-3.137 1.182 d 1.228-1.149 S = singlet, m = multiplet, d =doublet, dd = double doublet, q = quadruplet

2.10 Stability Study of Crystalline Genistein Potassium Salt Dihydrate

Sample stability was tested at 80° C. for 7 days and at 40° C./75 RH %for 7 days. Observations such as color change were noted after 7 daysand XRPD of samples were taken after 7 days to investigate any solidform change. FIG. 21 shows the XRPD patterns of samples of crystallinegenistein potassium salt dihydrate at 80° C. for 7 days and at 40° C./75RH % for 7 days. The 40° C./75 RH % study indicates that genisteinpotassium salt crystallizes to form genistein potassium salt dihydrate,Storing crystalline genistein potassium salt dihydrate at 80° C. over a7 day period has indicated crystallization to genistein potassium saltdihydrate.

2.11 Hydration Study of Crystalline Genistein Potassium Salt Dihydrate

Approximately 100 mg of crystalline genistein sodium salt dihydrate wasplaced in ca. 500 μL IPA/water mixtures (3%, 5% and 10%) at the waterlevel. Each mixture was agitated for ca. 48 hours at ambient temperatureand then filtered to recover the solid for XRPD and TGA studies. Asshown in FIG. 22, the hydration study revealed a hydrate consistent withcrystalline genistein potassium salt dihydrate.

2.12 Disproportionation Study of Crystalline Genistein Potassium SaltDihydrate

A sample of crystalline genistein potassium salt dihydrate was slurriedin distilled water for ca. 48 hours and then checked by XRPD fordisproportionation. The pH of the supernatant was also measured using aCorning 240 pH meter. No signs of disproportionation were observed. ThepH of the supernatant liquid was 7.3 indicating no disproportionation.

Example 3 Crystalline Genistein Calcium Salt

3.1 Preparation of Crystalline Genistein Calcium Salt

Approximately 25 mg of genistein was placed in the same vessel as ca. 7mg of solid calcium hydroxide, To the solid mixture, 500 μL of IPA/Water(50:50) was added and the mixture shaken at ambient temperature for ca.24 hours. Subsequent to stirring, the slurry was then temperature cycled(40° C. to ambient temperature, in 4 hour periods) with shaking for ca.72 hours. The solid was then isolated by filtration and allowed to dryat ambient temperature for ca. 24 hours.

3.2 Characterization of Crystalline Genistein Calcium Salt

FIG. 23 shows the XRPD pattern of the crystalline genistein calciumsalt. The peaks in the XRPD pattern at an experimental °2θ±0.2°2θ arelisted in Table 18. The entire list of peaks, or a subset thereof, maybe sufficient to characterize crystalline genistein calcium salt. Onesubset of peaks that, individually or in combination, may be used tocharacterize crystalline genistein calcium salt from FIG. 23 includes8.0, 15.3, 25.1, and 25.6°2θ±0.2°2θ. The TGA of crystalline genisteincalcium salt is shown in FIG. 24. The PIM image of the crystallinegenistein calcium salt showed needle-shaped crystals.

TABLE 18 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 5.4 16.2 21.828.0 11.0 98.01 9.3 9.5 17.32 15.3 5.8 100.00 15.5 5.7 17.27 16.0 5.631.04 16.3 5.5 24.40 16.8 5.3 6.33 18.5 4.8 17.43 19.0 4.7 6.23 20.3 4.416.69 21.5 4.1 5.44 22.5 3.9 7.30 22.7 3.9 14.06 23.0 3.9 7.86 24.1 3.76.61 25.1 3.5 37.75 25.6 3.5 72.44 26.1 3.4 15.90 27.8 3.2 20.14 29.23.1 23.14 29.5 3.0 34.31 30.0 3.0 15.01 33.5 2.7 19.69

Example 4 Crystalline Genistein Magnesium Salt, 1 Equivalent Preparation

4.1 Preparation of Crystalline Genistein Magnesium Salt, 1 Equivalent

Approximately 25 mg of genistein was placed in the same vessel as ca.5.5 mg of solid magnesium hydroxide. To the solid mixture, 500 μL ofIPA/Water (50:50) was added and the mixture shaken at ambienttemperature for ca. 24 hours. Subsequent to stirring, the slurry wasthen temperature cycled (40° C. to ambient temperature, in 4 hourperiods) with shaking for ca. 72 hours. The solid was then isolated byfiltration and allowed to dry at ambient temperature for ca. 24 hours.

4.2 Characterization of Crystalline Genistein Magnesium Salt, 1Equivalent

FIG. 25 shows the XRPD pattern of the crystalline genistein magnesiumsalt from the 1 equivalent preparation. The peaks in the XRPD pattern atan experimental °2θ±0.2°2θ are listed in Table 19. The entire list ofpeaks, or a subset thereof, may be sufficient to characterizecrystalline genistein magnesium salt. One subset of peaks that,individually or in combination, may be used to characterize crystallinegenistein magnesium salt from FIG. 25 includes 9.0, 18.6, 23.7, 25.7,and 38.0°2θ±0.2°2θ. The TGA of crystalline genistein magnesium salt, 1equivalent preparation, is shown in FIG. 26. The PLM image ofcrystalline genistein magnesium salt, 1 equivalent preparation, showedthe genistein magnesium salt to be crystalline.

TABLE 19 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 5.8 15.2 10.519.0 9.8 65.09 13.9 6.4 49.78 18.6 4.8 84.23 20.8 4.3 17.43 23.7 3.881.78 25.7 3.5 57.42 27.3 3.3 15.2 29.8 3.0 15.46 34.9 2.6 4.44 38.0 2.4100

Example 5 Crystalline Genistein Magnesium Salt, 2 Equivalent Preparation

5.1 Preparation of Crystalline Genistein Magnesium Salt, 2 Equivalents

Approximately 25 mg of genistein was placed in the same vessel as ca. 11mg of solid magnesium hydroxide. To the solid mixture, 500 μL ofIPA/Water (50:50) was added and the mixture shaken at ambienttemperature for ca. 24 hours, Subsequent to stirring, the slurry wasthen temperature cycled (40° C. to ambient temperature, in 4 hourperiods) with shaking for ca. 72 hours. The solid was then isolated byfiltration and allowed to dry at ambient temperature for ca. 24 hours.

5.2 Characterization of Crystalline Genistein Magnesium Salt, 2Equivalent Preparation

FIG. 27 shows the XRPD pattern of the crystalline genistein magnesiumsalt, 2 equivalent preparation. The peaks in the XRPD pattern at anexperimental °2θ±0.2°2θ are listed in Table 20. The entire list ofpeaks, or a subset thereof, may be sufficient to characterizecrystalline genistein magnesium salt. The TGA trace of crystallinegenistein magnesium salt, 2 equivalents, is shown in FIG. 28.

TABLE 20 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 9.0 9.8 30.2813.9 6.4 20.73 18.6 4.8 81.86 20.8 4.3 12.63 23.7 3.8 38.11 25.7 3.528.52 27.3 3.3 8.99 29.7 3.0 9.05 38.0 2.4 100

The similar XRPD patterns and TGA traces for the crystalline genisteinmagnesium salt from both the 1 equivalent preparation and the 2equivalent preparation suggests that the same crystalline genisteinmagnesium salt is obtained from both preparations. One subset of XRPDpeaks that, individually or in combination, may be used to characterizecrystalline genistein magnesium salt includes 9.0, 18.6, 23.7, 25.7, and38.0°2θ±0.2°2 θ.

Example 6 Crystalline Genistein L-Lysine Salt

6.1 Preparation of Crystalline Genistein L-Lysine Salt

Approximately 25 mg of genistein was placed in the same vessel as cu. 15mg of solid L-lysine monohydrate, To the solid mixture, 500 μL of eitherIPA or of toluene was added and the mixture shaken at ambienttemperature for ca. 24 hours. Subsequent to stirring, the slurry wasthen temperature cycled (40° C. to ambient temperature, in 4 hourperiods) with shaking for ca. 72 hours. The solid was then isolated byfiltration and allowed to dry at ambient temperature for ca. 24 hours.

6.2 Characterization of Crystalline Genistein L-Lysine Salt/GenisteinMixtures

Samples of crystalline genistein L-lysine salt from toluene and IPA wereanalyzed by XRPD and the XRPD patterns shown in FIGS. 30 and 31 weregenerated. The XRPD pattern for crystalline genistein is also shownbelow. As indicated by the XRPD, both methods produced mixtures ofgenistein and genistein L-lysine salt. FIG. 29 shows the XRPD pattern ofthe crystalline genistein. The peaks in the XRPD pattern of FIG. 29 atan experimental °2θ±0.2°2θ are listed in Table 21.

TABLE 21 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 7.5 11.7 81.8112.2 7.3 15.94 12.8 6.9 52.19 14.3 6.2 27.23 14.6 6.1 28.38 14.8 6.030.72 15.0 5.9 11.15 16.0 5.5 52.69 16.6 5.3 15.61 18.1 4.9 100 19.2 4.621.11 21.0 4.2 47.41 22.4 4.0 12.28 22.6 3.9 10.2 23.2 3.8 23.76 23.63.8 9.17 24.8 3.6 51.75 25.1 3.6 33.66 25.6 3.5 24.89 26.3 3.4 81.1226.7 3.3 23.35 27.0 3.3 10.41 27.4 3.3 56.24 28.7 3.1 56.15 29.4 3.030.44 33.6 2.7 14.28 35.9 2.5 7.63 39.5 2.3 13.43 40.1 2.3 7.68

6.3 Characterization of Crystalline Genistein L-Lysine Salt from Toluene

FIG. 30 shows the XRPD pattern of the crystalline genistein L-lysinesalt from toluene. The peaks in the XRPD pattern at an experimental°2θ±0.2°2θ are listed in Table 22.

TABLE 22 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 5.2 17.1 73.487.5 11.7 94.46 10.2 8.7 12.47 12.2 7.2 23.99 12.8 6.9 57.82 14.3 6.228.25 14.6 6.1 27.59 14.8 6.0 32.21 15.0 5.9 17.49 16.0 5.5 45.2 16.65.3 19.53 18.1 4.9 77.67 18.6 4.8 46.56 19.2 4.6 28.29 19.7 4.5 34.9820.6 4.3 20.97 21.0 4.2 17.82 21.2 4.2 11.09 22.4 4.0 17.25 22.6 3.912.61 23.2 3.8 21.8 23.6 3.8 12.36 24.8 3.6 25.82 25.1 3.6 27.26 25.63.5 30.47 26.3 3.4 100 26.7 3.3 23.56 27.0 3.3 14.34 27.4 3.3 50.01 27.93.2 6.19 28.7 3.1 46.23 29.4 3.0 40.47 32.9 2.7 9.67 33.6 2.7 12.33 36.02.5 7.24 36.3 2.5 7.15 39.5 2.3 5.12 40.0 2.3 6.87

6.4 Characterization of Crystalline Genistein L-Lysine Salt from IPA

FIG. 31 shows the XRPD pattern of the crystalline genistein L-lysinesalt from IPA. The peaks in the XRPD pattern at an experimental°2θ±0.2°2θ are listed in Table 23. The TGA of the crystalline genisteinL-lysine/genistein mixture is shown in FIG. 32. The PLM image of thegenistein L-lysine/genistein mixture from IPA snowed crystallinematerial as did the PLM image of the crystalline mixture from toluene.

TABLE 23 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 5.2 17.1 31.937.5 11.7 73.22 12.2 7.2 16.11 12.8 6.9 50.35 14.3 6.2 19.6 14.6 6.124.47 14.8 6.0 30.13 16.0 5.5 43.55 16.6 5.3 12.52 18.1 4.9 100 18.6 4.828.99 19.2 4.6 20.18 19.7 4.5 24.98 20.6 4.3 18.1 21.0 4.2 14.36 22.44.0 9.6 23.2 3.8 22.54 23.6 3.8 9.93 24.8 3.6 16.59 25.1 3.6 22.5 25.63.5 25.73 26.3 3.4 70.86 26.7 3.3 17.84 27.0 3.3 12.67 27.4 3.3 43.3428.7 3.1 38.7 29.4 3.0 28.79 32.9 2.7 6.17 33.4 2.7 6.94 36.3 2.5 7 40.02.3 7.5

The similar XRPD patterns for the crystalline genistein L-lysine saltfrom both isopropanol and toluene suggests that the same crystallinegenistein L-lysine salt is obtained from both preparations. The entirelist of peaks from Table 21 or 22, or a subset thereof, may besufficient to characterize crystalline genistein L-lysine salt. Bycomparing the crystalline genistein L-lysine salt XRPD patterns in FIGS.30 and 31 with that of crystalline genistein in FIG. 29, one subset ofpeaks that, individually or in combination, may be used to characterizethe crystalline genistein L-lysine salt includes 5.2, 18.6, 19.7, 20.6and 21.0°2θ±0.2°2θ.

Example 7 Crystalline Genistein N-methylglucamine (Meglumine) Salt

7.1 Preparation of Crystalline Genistein N-methylglocamine Salt

Approximately 25 mg of genistein was placed in the same vessel as ca. 20mg of solid N-methylglucamine. To the solid mixture, 500 μL of acetonewas added and the mixture shaken at ambient temperature for ca. 24hours. Subsequent to stirring, the slurry was then temperature cycled(40° C. to ambient temperature, in 4 hour periods) with shaking for ca.72 hours. The solid was then isolated by filtration and allowed to dryat ambient temperature for ca. 24 hours.

7.2 Characterization of Crystalline Genistein N-methylglucamine Salt

FIG. 33 shows the XRPD pattern of the crystalline genisteinN-methylglucamine salt. The peaks in the XRPD pattern at an experimental°2θ±0.2°2θ are listed in Table 24. The entire list of peaks, or a subsetthereof, may be sufficient to characterize crystalline genisteinN-methylglucamine salt. One subset of peaks that, individually or incombination, may be used to characterize crystalline genisteinN-methylgiucamine salt from FIG. 33 includes 7.5, 7.8, 12.3, 14.8, 16.5,17.1, 17.6, 18.8, 19.4, 20.0, 20.8, and 29.1°2θ±0.2°2θ. A preferredsubset includes peaks at 12.3, 14.8, 17.6, and 19.4°2θ±0.2°2θ.

TABLE 24 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 7.5 11.7 8.647.8 11.4 8.13 12.3 7.2 13.7 12.8 6.9 3.35 14.0 6.3 17.93 14.8 6.0 4.1116.0 5.5 4.38 16.5 5.4 8.33 17.1 5.2 7.93 17.6 5.0 47.95 18.1 4.9 5.518.8 4.7 21.35 19.4 4.6 100 20.0 4.4 7.27 20.8 4.3 12.4 26.1 3.4 6.8726.9 3.3 8.11 27.4 3.3 7.6 29.1 3.1 11.01 36.6 2.5 9.51

Example 8 Crystalline Genistein N-ethylglucamine (eglumine) Salt

8.1 Preparation of Crystalline Genistein N-ethylglucamine (eglumine)Salt

Approximately 25 mg of genistein was placed in the same vessel as ca. 19mg of solid N-ethylgiucamine. To the solid mixture, 500 μL of acetone orof IPA was added and the mixture shaken at ambient temperature for ca.24 hours. Subsequent to stirring, the slurry was then temperature cycled(40′C to ambient temperature, in 4 hour periods) with shaking for ca. 72hours. The solid was then isolated by filtration and allowed to dry atambient temperature for ca. 24 hours.

8.2 Characterization of Crystalline Genistein N-ethylglucamine(Eglumine) Salt

The sample of crystalline genistein n-ethylglucamine (eglumine) salt,prepared above, was analyzed by XRPD and the patterns shown in FIGS. 34and 35 generated. An unstable crystalline salt was identified both fromacetone and IPA. FIG. 34 shows the XRPD pattern of the crystallinegenistein N-ethylglucamine (eglumine) salt from acetone. The peaks inthe XRPD pattern at an experimental °2θ±0.2°2θ are listed in Table 25.FIG. 35 shows the XRPD pattern of the crystalline genisteinN-ethylglucamine (eglumine) salt from IPA. The peaks in the XRPD patternat an experimental °2θ±0.2°2θ are listed in Table 26 The entire list ofpeaks in either Table, or a subset thereof, may be sufficient tocharacterize crystalline genistein N-ethylglucamine (eglumine) salt. Onesubset of peaks that, individually or in combination, may be used tocharacterize crystalline genistein N-ethylglucamine (eglumine) saltbased on FIGS. 34 and 35 includes 7.4, 12.7, 14.7, 16.0, 18.1, 19.0,19.2, 21.7, 22.1, and 26.3°2θ±0.2°2θ. A preferred subset of peaksincludes 7.4, 12.7, 14.7, 16.0, 18.1, and 26.3°2θ±0.2°2θ. The TGA traceof crystalline genistein n-ethylglucamine salt from acetone is shown inFIG. 36. The PLM image of genistein n-ethylglucamine salt from acetoneshowed the material to be crystalline.

TABLE 25 Pos. [°2 θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 7.4 12.0 1007.5 11.7 14.48 12.7 6.9 25.42 14.7 6.0 34.98 16.0 5.5 8.49 18.1 4.9 9.3219.0 4.7 26.42 19.5 4.6 43.35 21.7 4.1 72.24 22.1 4.1 22.61 24.1 3.76.38 25.2 3.5 10.46 26.3 3.4 19.16 26.6 3.3 7.45 27.4 3.3 7.06 28.3 3.26.07 28.7 3.1 8.56 29.3 3.1 9.52 30.2 3.0 11.72 32.0 2.8 7.13 32.1 2.86.66 33.7 2.7 4.59 35.3 2.5 7 42.0 2.1 4.96

TABLE 26 Pos. [°2θ ± d-spacing Rel. Int. 0.2 °2θ] [Å] [%] 7.4 12.0 1007.5 11.7 49.75 12.8 6.9 37.57 14.7 6.0 42.98 16.0 5.5 19.76 16.6 5.38.05 18.1 4.9 27.66 19.0 4.7 32.79 19.2 4.6 12.86 19.4 4.6 48.27 21.74.1 80.33 22.1 4.0 24.33 23.2 3.8 8.12 24.8 3.6 7.52 25.0 3.6 14.01 25.23.5 15.58 25.6 3.5 14.97 26.3 3.4 84.13 26.7 3.3 27.05 27.4 3.3 24.3928.6 3.1 33.81 29.3 3.0 11.44 29.4 3.0 16.47 30.2 3.0 14.82 32.0 2.810.04 33.7 2.7 6.79 35.3 2.5 8.64 47.1 1.9 7.99

Example 9 Crystalline Genistein Diethylamine Salt

9.1 Preparation of Crystalline Genistein Diethylamine Salt

A stock solution of genistein in THF (520.2 mg in 19.25 of THF) anddiethylamine in THF:ETOH (1:1) was prepared. The stock solutions ofgenistein and diethylamine were added together in stoichiometric amountsand the solution was filtered through 0.2 μm nylon filters into cleanvials and allowed to evaporate under ambient conditions.

9.2 Characterization of Crystalline Genistein Diethylamine Salt

The solid material isolated above was subjected to XRPD analysis usingan Inel XRG-3000 diffractometer equipped with a CPS (Curved PositionSensitive) detector with a 2θ range of 120°; a Shimadzu XRD-6000 X-raypowder diffractometer using Cu Kα radiation and a Bruker D-8 Discoverdiffractometer equipped with Bruker's General Area Diffraction DetectionSystem (GADDS, v. 4.1.19). The specific acquisition parameters arelisted on the pattern of each sample in the data section. FIG. 37 showsthe XRPD pattern of the crystalline genistein diethylamine salt. Thepeaks in the XRPD pattern at an experimental °2θ±0.2°2θ are listed inTable 27. The entire list of peaks, or a subset thereof, may besufficient to characterize crystalline genistein diethylamine salt. Onesubset of peaks that, individually or in combination, may be used tocharacterize crystalline genistein diethylamine salt from FIG. 37includes 7.4, 8.2, 15.3, 25.3, and 28.4°2θ±0.1°2θ.

TABLE 27 °2θ d space (Å) Intensity (%)  6.7 ± 0.1 13.215 ± 0.200  30 7.4 ± 0.1 11.922 ± 0.163  50  8.2 ± 0.1 10.768 ± 0.133  41  9.1 ± 0.19.671 ± 0.107 21 10.0 ± 0.1 8.837 ± 0.089 29 11.2 ± 0.1 7.910 ± 0.071 2513.4 ± 0.1 6.608 ± 0.049 22 14.9 ± 0.1 5.937 ± 0.040 36 15.3 ± 0.1 5.803± 0.038 73 17.1 ± 0.1 5.185 ± 0.030 20 18.0 ± 0.1 4.937 ± 0.027 19 18.3± 0.1 4.844 ± 0.026 18 20.1 ± 0.1 4.430 ± 0.022 18 21.1 ± 0.1 4.207 ±0.020 16 22.4 ± 0.1 3.969 ± 0.018 20 23.4 ± 0.1 3.796 ± 0.016 15 24.7 ±0.1 3.607 ± 0.014 33 25.3 ± 0.1 3.524 ± 0.014 100 26.5 ± 0.1 3.370 ±0.013 22 27.0 ± 0.1 3.297 ± 0.012 18 27.8 ± 0.1 3.213 ± 0.011 23 28.4 ±0.1 3.143 ± 0.011 43 28.7 ± 0.1 3.110 ± 0.011 30 29.3 ± 0.1 3.049 ±0.010 18

Example 10 Crystalline Genistein Monohydrate

10.1 Preparation of Crystalline Genistein Monohydrate

A stock solution of genistein was prepared in THF (472 mg in 17.47 mL ofTHF). Genistein stock solution (1 mL) was added to a glass vial,followed by 1 mL of D-glucuronic acid solution (84.1 mg in 4.33 ofwater). The solution was allowed to evaporate under ambient conditions.Solids were isolated after 1 day by decanting the remaining solution andwere then blotted dry with filter paper.

10.2 Characterization of Crystalline Genistein Monohydrate

XRPD analysis of the crystalline genistein monohydrate sample wasperformed using an Inel XRG-3000 diffractometer equipped with a CPS(Curved Position Sensitive) detector with a 2θ range of 120°; a ShimadzuXRD-6000 X-ray powder diffractometer using Cu Kα radiation; and a BrukerD-8 Discover diffractometer equipped with Bruker's General AreaDiffraction Detection System (GADDS, v. 4.1.19). FIG. 38 shows the XRPDpattern of the crystalline genistein monohydrate. The peaks in the XRPDpattern at an experimental °2θ±0.2°2θ are listed in Table 28. The entirelist of peaks, or a subset thereof, may be sufficient to characterizecrystalline genistein monohydrate. One subset of peaks that,individually or in combination, may be used to characterize crystallinegenistein monohydrate from FIG. 38 includes 9.0, 11.3, 13.4, 14.8, 23.1,25.0, 26.8, and 28.5 2θ±0.1°2θ.

TABLE 28 °2θ d space (Å) Intensity (%)  9.0 ± 0.1 9.819 ± 0.110 65 11.3± 0.1 7.862 ± 0.070 52 13.0 ± 0.1 6.800 ± 0.052 36 13.4 ± 0.1 6.591 ±0.049 100 14.1 ± 0.1 6.269 ± 0.044 31 14.8 ± 0.1 5.992 ± 0.041 50 18.3 ±0.1 4.844 ± 0.026 36 22.1 ± 0.1 4.024 ± 0.018 40 22.5 ± 0.1 3.951 ±0.017 32 23.1 ± 0.1 3.857 ± 0.017 70 23.8 ± 0.1 3.741 ± 0.016 36 24.0 ±0.1 3.709 ± 0.015 34 24.7 ± 0.1 3.612 ± 0.014 34 25.0 ± 0.1 3.562 ±0.014 55 25.9 ± 0.1 3.436 ± 0.013 45 26.8 ± 0.1 3.331 ± 0.012 61 27.8 ±0.1 3.209 ± 0.011 25 28.5 ± 0.1 3.136 ± 0.011 54 29.0 ± 0.1 3.081 ±0.010 26

The thermogravimetric analysis (TGA) of crystalline genisteinmonohydrate was performed using a TA Instruments 2950 thermogravimetricanalyzer. FIG. 39 shows the TGA trace of the crystalline genisteinmonohydrate sample. Thermogravimetric analysis indicated that the samplecontained 6% by weight of volatile component, which is equivalent to amonohydrate.

The invention claimed is:
 1. A genistein composition selected from:genistein potassium dihydrate, a genistein calcium salt, a genisteinmagnesium salt, a genistein L-lysine salt, a genistein N-methylglucaminesalt, a genistein N-ethylglucamine salt, a genistein diethylamine salt,and crystalline genistein monohydrate.
 2. The genistein composition ofclaim 1, wherein the composition is genistein potassium dihydrate ofclaim 19 in the form of crystalline genistein potassium salt dihydratecharacterized by an XRPD pattern having peaks at 11.6, 14.5, 24.5, 25.2and at least two of the three peaks 27.6, 28.0, and 28.4 ° 2θ±0.2° 2θ.3. The genistein composition of claim 1 wherein the composition is acrystalline genistein calcium salt, a crystalline genistein magnesiumsalt, a crystalline genistein L-lysine salt, a crystalline genisteinN-methylglucamine salt, a crystalline genistein N-ethylglucamine salt,or a crystalline genistein diethylamine salt.
 4. The genisteincomposition of claim 1, wherein the composition is crystalline genisteinmonohydrate.
 5. A therapeutic composition comprising a therapeuticallyeffective amount of the genistein composition of claim 1 and at leastone pharmaceutically acceptable carrier.
 6. A method of treating chronicinflammation or inflammatory diseases comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of the therapeutic composition of claim
 5. 7. A method fortreating chronic inflammation or inflammatory diseases comprising thestep of administering to a patient in need thereof a therapeuticallyeffective amount of the genistein composition of claim
 1. 8. A methodfor treating transthyretin amyloidosis comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of the therapeutic composition of claim
 5. 9. A method fortreating transthyretin amyloidosis comprising the step of administeringto a patient in need thereof a therapeutically effective amount of thegenistein composition of claim
 1. 10. A method for treating cysticfibrosis comprising the step of administering to a patient in needthereof a therapeutically effective amount of the therapeuticcomposition according to claim
 5. 11. A method for treating cysticfibroses comprising the step of administering to a patient in needthereof a therapeutically effective amount of the genistein compositionof claim
 1. 12. A method for treating infection comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of the therapeutic composition according to claim
 5. 13. A methodfor treating infection comprising the step of administering to a patientin need thereof a therapeutically effective amount of the genisteincomposition of claim
 1. 14. The genistein composition of claim 3,wherein the crystalline genistein diethylamine salt is characterized byan XRPD pattern of FIG.
 37. 15. The genistein composition of claim 3,wherein the crystalline genistein diethylamine salt is characterized byan XRPD pattern having peaks at 7.4, 8.2, 15.3, 25.3, and 28.4 ° 2θ±0.1°2θ.
 16. A method of arresting or suppressing the development of clinicalsymptoms related to cancer comprising the step of administering to apatient in need thereof a therapeutically effective amount of thetherapeutic composition of claim
 5. 17. A method of arresting orsuppressing the development of clinical symptoms related to cancercomprising the step of administering to a patient in need thereof atherapeutically effective amount of the genistein composition ofclaim
 1. 18. The method of claim 16, wherein said cancer is selectedfrom the group consisting of colorectal cancer, gastric cancer,esophageal cancer, breast cancer, lung cancer, prostate cancer, bladdercancer, brain cancer, renal cancer, ovarian cancer, liver cancer, skincancer, thyroid cancer, pancreatic cancer, leukemia, and lymphoma. 19.The method of claim 17, wherein said cancer is selected from the groupconsisting of colorectal cancer, gastric cancer, esophageal cancer,breast cancer, lung cancer, prostate cancer, bladder cancer, braincancer, renal cancer, ovarian cancer, liver cancer, skin cancer, thyroidcancer, pancreatic cancer, leukemia, and lymphoma.